CN1355703A - Therapeutical agent - Google Patents

Abstract

Remedies or preventives for diseases with a need for a growth factor production-inducing effect, characterized by containing member(s) selected from the group consisting of acidic polysaccharides and degradation products thereof, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols and salts thereof each having an effect of inducing the production of growth factor; foods, drinks or feeds for inducing the production of growth factor; cosmetics for inducing the production of growth factor; and growth factor production regulators.

Description

Therapeutic agent

Technical field

The present invention relates to have the purposes of the acid sugar chemical compound of physiologically active as medicine, food, beverage, feedstuff or cosmetics.

Background technology

As the acidic polysaccharose by the Sargassum origin, known have the sulfuric acid rhamnosan (ラ system Na Application) that contains the green algae origin, the sulfated galactan of red algae origin, sulfated polysaccharides such as polysaccharide of the sulphation fucose of Brown algae origin.For example, fucoidan is the polysaccharide of the sulfur-bearing acidify fucose that contains in Brown algae, echinoderm etc., is to contain the sulphation fucose as the material that constitutes sugar.In addition, shark cartilage etc. also contains sulfated polysaccharides.

As the physiological action of sulfated polysaccharides, fucoidan for example, known have a cancer proliferation inhibition activity, and it is active that cancerometastasis suppresses, anticoagulant active, therefore antiviral activities etc. expect its exploitation as medicinal usage.

As having the inducing material of hepatocyte proliferation factor, the known low molecular weight heparin (spy opens flat 6-312941 communique) that heparin, Heparan sulfate, mean molecule quantity 4400～5600 are arranged, but, about other sulfated polysaccharide, for example fucoidan, synthetic sulfated polysaccharides etc. do not produce the report that inducing action is arranged to somatomedin.

Disclosure of an invention

The present invention has found various acid sugar chemical compounds, acidic polysaccharose for example, new physiological action as fucoidan etc., its objective is to provide and utilize various acid sugar chemical compounds, acidic polysaccharose for example, produce inducing action, particularly hepatocyte proliferation factor as the somatomedin of fucoidan etc. and produce medicine, food, beverage, feedstuff or the cosmetics that inducing action, Insulin-Like multiplicaiton factor produce inducing action or nerve growth factor production inducing action.

If general introduction the present invention, the 1st invention of the present invention need to relate to somatomedin to produce inductive treatment of diseases agent or preventive, it is characterized in that containing be selected from have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and their material of salt (condition is, except the heparin, Heparan sulfate) as effective ingredient.

The 2nd invention of the present invention relate to contain be selected from have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and the somatomedin that forms of their material of salt produce and induce with food, beverage (hereinafter referred to as the diet product) or feedstuff.

The 3rd invention of the present invention relate to contain be selected from have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and the somatomedin that forms of their material of salt produce to induce and apply some make up.

The 4th invention of the present invention relate to contain be selected from acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and the somatomedin that forms of their material of salt produce regulator.

In the present invention, as having the inducing acidic polysaccharose of somatomedin, preferably be example with the sulfated polysaccharides, can suitably use the sulfated polysaccharides of algae origin as sulfated polysaccharides; The sulfated polysaccharides of animal origin, the sulfated polysaccharides of echinoderm origin for example is as the sulfated polysaccharides of Stichopus japonicus origin; The sulfated polysaccharides of Fish origin, for example sulfated polysaccharides of shark cartilage origin; The sulfated polysaccharides of microorganism origin; The sulfated polysaccharides of plant origin, for example sulfated polysaccharides of Radix Artemisia ordosicae origin; Synthetic sulfated polysaccharides.

In addition, as sulfated polysaccharides, can suitably use and contain sulfuric acid rhamnosan, sulfated galactan, or the polysaccharide of sulphation fucose with the inducing algae origin of somatomedin.Can suitably use for example dextran sulfate sodium as synthetic sulfated polysaccharide, sulphation starch, sulphation カ one De ラ Application, sulphation pectin etc. are by the high sulphation sulfated polysaccharides that the further sulphation of sulfated polysaccharides is obtained.In addition, can suitably use fucoidan as the polysaccharide that contains the sulphation fucose.As acidic oligosaccharide preferably sulfuric acid oligosaccharide, can use for example sulphation maltose, the sulphation lactose, sulphation sucrose, sulphation trehalose, sulphation lactulose, the sulphation 6-(.alpha.-D-galactosido)-D-glucose., sulphation cellobiose, sulphation dextrinose, the sulphation turanose, sulphation パ ラ チ ノ one ス, sulphation maltotriose, Maltohexaose sulfate, sulphation Fructus Hordei Germinatus seven sugar, sulphation dodecyl MALTOHAXAOASE, chemical compound shown in chemical compound shown in the following formula (I) or the following formula (II).

(R represents OH or OSO in the formula ₃H.)

(R represents OH or OSO in the formula ₃H.)

As acid monosaccharide, preferably sulfuric acid monosaccharide for example can use, Glucose sulfate, sulphation galactose, sulphation xylose, sulphation 2-deoxyglucose, sulphation talose and sulphation mannose.Can use the hydrosulphate of sugar alcohol, for example sulphation glucitol as acid sugar alcohol.These sulfated oligosaccharides, sulphation monosaccharide, sulphation sugar alcohol can prepare with general synthetic method.As long as these sulfated oligosaccharides, sulphation monosaccharide, sulphation sugar alcohol show that somatomedin produces inducing action, has no particular limits the sulfate position in these sugar compounds, sulfate quantity.

Also can use the inducing acidic polysaccharose degradation product of somatomedin in the present invention.Also comprise in this degradation product and have depolymerized heparin thing, the Heparan sulfate degradation product of the inducing molecular weight of somatomedin below 4000.

Cited material can use separately also and can mix use more than 2 kinds in aforementioned acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, the acid sugar alcohol, in addition, also can suitably use their salt.

In the present invention, can enumerate hepatocyte proliferation factor, Insulin-Like multiplicaiton factor and nerve growth factor as somatomedin.

Therapeutic agent or preventive in the 1st invention of the present invention, food, beverage or the feedstuff of the 2nd invention, in the cosmetics of the 3rd invention, but the inducing material of somatomedin that can further contain Synergistic acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt, can enumerate as this material and to be selected from cytokine class, prostaglandins contains the chemical compound of cyclopentenes ring, the material in minoxidil and the carpronium chloride.

In addition, food, beverage or the feedstuff of the 2nd invention of the present invention preferably have food, beverage or feedstuff that hepatocyte proliferation factor produces inducing action, Insulin-Like multiplicaiton factor generation inducing action or nerve growth factor production inducing action.

In addition, the cosmetics of the 3rd invention of the present invention preferably have the cosmetics that hepatocyte proliferation factor produces inducing action, Insulin-Like multiplicaiton factor generation inducing action or nerve growth factor production inducing action.

As the cosmetics of the 3rd invention of the present invention, can enumerate lotion class, emulsion class, cream kind, face film agent, baths, the agent of washing one's face is bathed with soap or is bathed with detergent etc.

In addition, " be selected from acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and their material of salt " among the present invention abbreviates " effective ingredient " in this manual sometimes as.

Brief description of drawings

Fig. 1 is the figure that shows the elution curve of fucoidan on the DEAE-CellulofineA-800 post of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin.

Fig. 2 is the inspection amount line chart that metabisulfite solution is shown sulfuric acid content as standard reagent.

The best mode that invention is implemented

In the present invention, what is called has the inducing acidic polysaccharose of somatomedin, can be any inducing acidic polysaccharose of somatomedin that has, this is had no particular limits, can suitably use acidic polysaccharoses such as alginic acid, pectin, pectic acid, hyaluronic acid; Chondroitin sulfate, keratan sulfate, sulfated polysaccharides such as dermatan sulfate; The sulfated polysaccharides of animal origin, for example, the sulfated polysaccharides of echinoderm origin; The sulfated polysaccharides of Fish origin, for example sulfated polysaccharides of shark cartilage origin; The plant origin arrives sulfated polysaccharides, for example, and the sulfated polysaccharides of Radix Artemisia ordosicae origin, the sulfated polysaccharide of Fructus Momordicae charantiae origin, the sulfated polysaccharide of Aloe origin, the sulfated polysaccharide of chrysanthemum leaf origin; The sulfated polysaccharide of microorganism origin, the sulfated polysaccharide of Chlorella origin for example, the sulfated polysaccharide of Spirullina origin; The sulfated polysaccharide of algae origin etc.

Can use the sulfuric acid rhamnosan of algae origin as the sulfated polysaccharide of algae origin, the sulfated galactan of red algae origin, Eucheuma gelatinosum for example, the river hedge belongs to, Macrocystis pyrifera (L.) Ag., プ テ ロ Network ラ デ ィ ァ カ ピ ラ セ ァ, carrageenin, agar class, agarose, agaropectin, metalloporphyrin, the polysaccharide that contains the sulphation fucose of Brown algae origin, for example, fucoidan, sulfated fucogalactan, sulphation rock algae glucomannan, glucose xylose fucan, サ Le ガ ッ サ Application, the Fructus Vitis viniferae mannogalactan, xylose rock algae glucosan, ァ ス コ Off ィ ラ Application, glucose gala fucosan, sulphation glucose fucosan etc.Particularly, fucoidan, sulfated fucogalactan, λ-carrageenin, chondroitin sulfate B, chondroitin sulfate D, alginic acid, agaropectin etc. can preferably use in the present invention.In addition, also can use the acidic polysaccharose of blue algae origin, the sulfated polysaccharides of Spirullina origin for example, the acidic polysaccharose of green algae origin, for example sulfated polysaccharides of Chlorella origin.Particularly, it is useful to the improvement of liver function that the sulfated polysaccharides of Spirullina origin produces inducing action by its hepatocyte proliferation factor, for example, can significantly improve the symptom of C type hepatitis.In addition, phosphorylated polysaccharide, for example nucleic acid is also included within the acidic polysaccharose of the present invention.

As the polysaccharide that contains the sulphation fucose that uses among the present invention, preferably can enumerate the fucoidan of aforementioned algae origin, but so long as have the sulphation fucose as constituent and have that somatomedin is inducing can, this is had no particular limits, can use echinoderm, for example the fucoidan of origin such as Stichopus japonicus, Hemicentrotus seu Strongylocentrotus, Asterias amurensis Lutken.

These can separately or mix more than 2 kinds and use.In addition,, this is had no particular limits, also can use the degradation product or the salt of these illustrated acidic polysaccharoses as long as the degradation product or the salt of these illustrated acidic polysaccharoses also show the growth factor-induced effect.

These acidic polysaccharoses can use the known method preparation respectively, and purification thing or this acidic polysaccharose contain thing all can be used in the present invention.Contain thing as acidic polysaccharose and preferably use the sulfated polysaccharides part, preferably use the sulfated polysaccharides part of algae origin, the sulfated polysaccharides part of shark cartilage origin as this part.In addition, contain the raw material algae of thing, can use Stichopus japonicus, shark cartilage etc. as sulfated polysaccharides.For example, Sargassums such as Laminariales, Chordariales, Fucales such as ガ go メ Thallus Laminariae (Thallus Eckloniae), Thallus Laminariae (Thallus Eckloniae), ト ロ ロ Thallus Laminariae (Thallus Eckloniae), Fucus Vesiculosus, Nemacystus decipiens (Sur.) Kuck genus, Okinawa Nemacystus decipiens (Sur.) Kuck genus, undaria, goose palm dish, Eisenia, Thallus Laminariae (Thallus Eckloniae) genus, Macrocystis (レ ッ ソ ニ ァ ニ グ レ セ Application ス), Ascophyllum nodosum are owing to contain the preferred fucoidan that uses among the present invention in a large number, therefore, be preferred as raw material.

As the synthetic sulfated polysaccharides that uses in the present invention, so long as have somatomedin inducing can, this is had no particular limits, preferably use so far sulfated polysaccharides as drug use.Can enumerate dextran sulfate sodium as this synthetic sulfated polysaccharides.This chemical compound is the sodium salt of the sulfuric ester that obtains of the part degradation product sulphation of the glucosan that will produce by Leuconostoc mesenteroides vanTieghem sucrose fermentation.

Also can use synthetic sulfated polysaccharides such as sulphation starch, sulphation カ one De ラ Application, sulphation pectin in addition in the present invention, preferably use high sulphation sulfated polysaccharides by further the sulfated polysaccharides sulphation being obtained.

Sulfate position to the sulfated polysaccharides that uses in the present invention has no particular limits, as long as it shows the long-living inducing action of somatomedin.2 that constitute sugar by Sulfated sulfated polysaccharides, fucoidan, and λ-carrageenin, chondroitin sulfate D, their degradation product can preferably use in the present invention.In addition, the sulfuric acid content (or sulphuric acid radix) of sulfated polysaccharides is had no particular limits, as long as there have somatomedin to produce inducing action to be just passable.In addition, the degradation product of acidic polysaccharose also comprises oligosaccharide, monosaccharide, can use oligosaccharide, monosaccharide with 2 sulfates in the present invention, fucose-2-sulphuric acid for example, glucose-2-sulphuric acid.These sulphation monosaccharide, sulfated oligosaccharide, sulfated polysaccharides can use general synthetic method preparation, and preparation product, purification thing also can be used for the present invention.Oligosaccharide among the present invention is meant the chemical compound that 2～10 monosaccharide form, and polysaccharide is meant the chemical compound that the monosaccharide more than 11 forms.

For example, fucoidan from the preparation of ガ go メ Thallus Laminariae (Thallus Eckloniae), the separable fucoidan (F-fucoidan) for containing the fucoidan (U-fucoidan) of glucuronic acid and not containing glucuronic acid of this fucoidan can use these fucoidans respectively as effective ingredient of the present invention.In addition, can prepare sulfated fucogalactan and use from ガ go メ Thallus Laminariae (Thallus Eckloniae).

In addition, can prepare agaropectin and use from agar.

After U-fucoidan and F-fucoidan can prepare fucoidan from ガ go メ Thallus Laminariae (Thallus Eckloniae), with separation such as anion exchange resin, surfactants.The U-fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin and the existence of F-fucoidan ratio are about 1: 2, the U-fucoidan contains fucose, mannose, galactose, glucuronic acid etc., sulfuric acid content is about 20%, the F-fucoidan contains fucose and galactose, sulfuric acid content is about 50%, the molecular weight of two materials is central distribution (the 18th sugared monograph set abstracts collection, 159 pages, 1996) with 200,000 all.

After for example the fucoidan solution from the preparation of ガ go メ Thallus Laminariae (Thallus Eckloniae) can being applied to DEAE-Cellulofine A-800 post, contain the E-test eluting of the buffer of NaCl by use, separate U-fucoidan and F-fucoidan.Show an example among Fig. 1.That is, Fig. 1 is the separation graph of expression U-fucoidan and F-fucoidan, and among the figure, the peak of front is the U-fucoidan, and the peak of back is the F-fucoidan.

In addition, the sulfated polysaccharides of stone stilbene dish origin for example, the river hedge belongs to the sulfated polysaccharides of origin, the sulfated polysaccharides of プ テ ロ Network ラ デ ィ ァ origin, the sulfated polysaccharides of other algae origin, the fucoidan of Fucus Vesiculosus origin, Nemacystus decipiens (Sur.) Kuck belongs to the fucoidan of origin, the Okinawa Nemacystus decipiens (Sur.) Kuck belongs to the fucoidan of origin, the fucoidan of undaria origin, the fucoidan of Macrocystis origin, the fucoidan of Ascophyllum nodosum origin, the fucoidan of other algae origin also can prepare with known method respectively, and uses in the present invention.

As the Stichopus japonicus that contains fucoidan, for example have and specially to open the Stichopus japonicus of putting down in writing in the flat 4-91027 communique, the method for putting down in writing in also available this communique prepares fucoidan from Stichopus japonicus.

In addition, of the present invention have an inducing acidic polysaccharose degradation product of somatomedin, the degradation product of sulfated polysaccharides, fucoidan for example, the preparation of known method such as available Enzymology method, chemical method, physical method, select the required inducing degradation product of somatomedin that has, and use.

In addition, degradation product is meant molecular weight that the acidic polysaccharose degraded as the degraded object is obtained preferably about 100,000～200, more preferably from about the material of 30,000～1000 scopes.

Preferred for preparation method as the acidic polysaccharose degradation product that uses in the present invention is the acid degradation method, can be by this acidic polysaccharose acid degradation preparation is had the inducing degradation product of somatomedin.

The acid degradation condition of the acidic polysaccharose that uses for the present invention has no particular limits, and has the inducing degradation product of somatomedin as long as can generate.

For example, with acidic polysaccharose dissolving or be suspended in the acidic aqueous solution etc., generate degradation product of the present invention by reaction.In addition, can be by when reacting, the required time of degradation product of the present invention that generates is shortened in heating.

Kind for the acid of acidic polysaccharose being dissolved or suspending has no particular limits, and can use inorganic salts such as hydrochloric acid, sulphuric acid, nitric acid, citric acid, formic acid, acetic acid, lactic acid, organic acid such as ascorbic acid, or solid acid such as cation exchange resin, cation exchange fibre, cation exchange membrane.

Concentration for acid has no particular limits, can be at preferred 0.0001～5 equivalent, and more preferably the concentration about 0.01～1 equivalent is used.In addition, do not have specific restriction yet, can be set in preferred 0～200 ℃, more preferably 20～130 ℃ for reaction temperature.

In addition, also have no particular limits for the response time, can be set in preferred several seconds～a couple of days.The growing amount of the degradation product that the kind of acid and concentration, reaction temperature and response time use in can be according to the present invention, the degree of polymerization of degradation product are suitably selected.For example, when the degradation product of preparation fucoidan, can make organic acid such as citric acid, lactic acid, malic acid, at acid concentration 10mM～number M, 50～110 ℃ of heating-up temperatures, preferred 70～95 ℃, heat time heating time, the scope in several branches～24 hour was suitably selected, and can make degradation product of the present invention.Can enumerate the acid degradation thing of the fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin as the acid degradation thing of fucoidan, this degradation product can be used as has strong somatomedin generation inducing action, the particularly dietary fiber of the inducing new physiological function of hepatocyte proliferation factor use.

Degradation product of the present invention can produce somatomedin inducing action and carry out classification as index, for example, the acid degradation thing can be carried out molecular-weight gradation with stagings such as gel filtration, molecular-weight gradation films.

As the example of gel filtration, can use Cellulofine GCL-300, preparation example such as molecular weight surpass 25000, molecular weight 25000～surpass 10000, molecular weight 10000～surpass 5000, molecular weight 5000 is with inferior any molecular weight component; Can use CellulofineGCL-25 that the component of molecular weight below 5000 for example is prepared into molecular weight 5000～surpass 3000, molecular weight 3000～surpass 2000, molecular weight 2000～surpass 1000, molecular weight 1000～surpass 500, molecular weight 500 is with inferior any molecular weight component.

In addition, can use ultrafilter membrane at the industrial molecular-weight gradation that carries out.For example,, can prepare the component of molecular weight below 30000, use the FE-FUS-T653 of same companies system, can prepare the component of molecular weight below 6000 by using the FE10-FUS0382 of Daicel society system.In addition, can use nanometer film to obtain the component of molecular weight below 500,, can prepare the component of any molecular weight by with these gel filtrations, the combination of molecular-weight gradation method.

Spendable in the present invention have an inducing acidic polysaccharose degradation product of somatomedin, but for example as chemical compound shown in chemical compound shown in the degradation product enumerative (I) of fucoidan, the formula (II), these chemical compounds can use the international method of putting down in writing in 97/26896 and No. 99/41288 communique that disclose to prepare.In addition, having the sulfated polysaccharides of chemical compound repetitive construct shown in the formula (I) and oligosaccharide also can be used as and of the present inventionly has the inducing sulfated polysaccharides of somatomedin and use.

By aforementioned F-fucoidan is handled with the tip type sulfated polysaccharides digestive enzyme (F-fucoidan specificity digestive enzyme) of alternately monospore Pseudomonas SN-1009 (FERM BP-5747) generation, its degradation product of purification can obtain the chemical compound shown in the formula (I).About content, the position of sulfate in this chemical compound, can be from its degradation product the purification arbitrary substance.In addition, when containing the polymer of chemical compound shown in the formula (I) in this degradation product, can separate as required, purification.

By aforementioned U-fucoidan is handled with the tip type sulfated polysaccharides digestive enzyme (U-fucoidan specificity digestive enzyme) that Flavobacterium SA-0082 (FERM BP-5402) produces, its degradation product of purification can obtain the chemical compound shown in the formula (II).About content, the position of sulfate in this chemical compound, can be from its degradation product the purification arbitrary substance.In addition, when containing chemical compound shown in the formula (II) in this degradation product, can separate as required as the polymer of basic skeleton structure, purification.

Example as chemical compound shown in the formula (I) has chemical compound shown in the aftermentioned formula (VI).In addition, the example as chemical compound shown in the formula (II) has chemical compound shown in the aftermentioned formula (VII).

In addition, the fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin can be obtained the polymer of glucuronic acid and mannose by heat treated in the presence of organic acid, this polymer also can be used as the inducing acidic polysaccharose use of somatomedin that has of the present invention.In addition can be by adjusting heating condition, preparing the polymer of any degree of polymerization heat time heating time.

As having the inducing acidic polysaccharose of somatomedin in the present invention, comprise synthetic sulfated polysaccharides, can use the hydrosulphate of cellulose, starch, mannan, xylan, alginic acid, pectin, pectic acid, levan, arabinan, chitosan, pullulan, xyloglucan, glucosan, starch etc.In addition, also can use synthetic sulfated polysaccharides such as the nuclear of sulphuric acid furan for example polysaccharide, sulphuric acid furan xylan, sulphuric acid lentinan, カ one De ラ Application sulphuric acid, sulphuric acid pyrans mannan or have the synthetic sulphation alkyl polysaccharides such as sulphuric acid furan xylan of hexadecanoyl.Can be by sulfated polysaccharides or the further sulphation of its degradation product be prepared high Sulfated sulfated polysaccharides or high-sulfur acidified degradation thing.These sulfated polysaccharides, high Sulfated sulfated polysaccharides, high-sulfur acidified degradation thing can prepare with known method respectively, the also available known method preparation of its degradation product, and use in the present invention.In addition, can use commercially available dextran sulfate, Cellulose sulfate, also can use the salt of these synthetic sulfated polysaccharides etc.

As acidic oligosaccharide, preferably can enumerate sulfated oligosaccharide, in addition,, preferably can enumerate sulphation monosaccharide as acid monosaccharide, their object lesson can be enumerated material as hereinbefore.Sulfated oligosaccharide or sulphation monosaccharide can use corresponding oligosaccharide, monosaccharide as raw material, carry out the sulphation preparation with known method.In addition, also can use their salt.In addition, derivative of fatty acid of sulfated polysaccharides, sulfated oligosaccharide, sulphation monosaccharide etc. is also included within sulfated polysaccharides of the present invention, sulfated oligosaccharide, the sulphation monosaccharide.These can separately or mix more than 2 kinds and use.

Desired in the present invention somatomedin of inducing generation is meant to have the cell of the promotion active factor of growing up, this is had no particular limits, can enumerate hepatocyte proliferation factor (HGF), nerve growth factor (NGF), neurotrophic factor, epithelium growth factor, the somatomedin of milk origin, fibroblast growth factor, the fibroblast growth factor of brain origin, acid fibroblast growth factor, the somatomedin of platelet origin, platelet basic protein, connective tissu es activating peptides, Insulin-Like multiplicaiton factor (IGF), bacterium colony forms stimulating factor, erythropoietin, blood is fastened formin, the T cell growth factor, interleukin class (interleukin-22 for example, 3,4,5,7,9,11,15), Bcell growth factor, the factor of cartilage origin, the somatomedin of cartilage origin, the somatomedin of bone origin, the bone growth factor, endothelial cell growth factor (ECGF), the somatomedin of endotheliocyte origin, the somatomedin of eye origin, the somatomedin of testis origin, the somatomedin of foot-cells origin, the mammary gland stimulating factor, the somatomedin of spinal cord origin, the somatomedin of macrophage origin, the regeneration mesodermal growth factor, transform multiplicaiton factor-α, transform multiplicaiton factor-β, heparin associativity EGF sample multiplicaiton factor, ァ Application Off ィ レ グ リ Application, SDGF, β-tunicin, epidermis is regulated albumen (ェ ピ レ グ リ Application), neuregulin 1,2,3, the blood vessel endothelium multiplicaiton factor, neurotrophin, BDNF, NT-3, NT-4, NT-5, NT-6, NT-7, the neurotrophic factor of glial cell-line origin, stem cell factor, midkine, pleiotrophin, ephrin, angiogenin, nandrolone phenylpropionate, tumor necrosis factor, interferon etc.

Wherein, from prevention, the treatment of hepatopathy, the prevention of nervous system disease, treatment, the prevention of diabetes, the viewpoint of treatment consider that preferred use is selected from least a the generation as effective ingredient in the present invention of HGF, NGF, IGF and induces.

HGF shows the hepatocyte growth effect, and albumen synthesize facilitation, and bile blocks the improvement effect, and the effect of the nephropathy that causes of prophylactic agent etc.In addition, the mRNA of HGF is also synthetic at brain, kidney, lung etc., and it is the mesoblastema somatomedin that hepatocyte, renal tubular cell, epidermis cell etc. is had proliferation activity.Therefore, by the generation of the inducing hepatocyte proliferation factor, can carry out the treatment or the prevention of hepatitis, hepatitis gravis, acute severe hepatitis, liver cirrhosis and hepatic bile retardance, chronic nephritis, pneumonia, wound.

IGF has various physiological actions in various cells.By inducing the generation of IGF, can carry out the treatment or the prevention of II-type diabetes (non-insulin-dependent) or growth obstacle (cretinism).

NGF is existence or a function of keeping neurocyte, Concentraton gradient according to NGF can be with the endogenous somatomedin of neurocyte elongation, by inducing the generation of NGF, can carry out treatment that needs the nervous function reparative regeneration or prevention that senile dementia such as Alzheimer or peripheral nerve obstacle, cerebrovascular disorders, cerebroma, brain apicitis, degenerative disease of head injury, anaesthetic are poisoned and caused.In addition, therapeutic agent of the present invention or preventive show the generation inducing action of neurotrophic factor, further by therapeutic agent of the present invention or preventive generation inducing action to the NGF neurotrophic factor, it is at muscular dystrophy lateral sclerosis, medicine obstacle peripheral nerve obstacle, diabetic peripheral nerve obstacle is useful in the treatment of Alzheimer's disease, parkinson, sensory nerve obstacle, retinitis pigmentosa, macular degeneration disease etc., the prevention.

The acidic polysaccharose that uses among the present invention, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or their salt has somatomedin and produces inducing action, can be with these chemical compounds manufacturing needs somatomedin to produce as effective ingredient treatment of diseases agent or preventive.

The somatomedin that needs of the present invention produces inductive treatment of diseases agent or preventive, can itself and known pharmaceutical carrier be combined to form preparation with the material that is selected from the present invention the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol and their salt that use as effective ingredient.The manufacturing of said preparation generally is to mix with the aqueous or solid carrier that pharmaceutically allows being selected from the material of the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol and their salt that use in the present invention, and as required, add solvent, dispersant, emulsifying agent, buffer agent, stabilizing agent, excipient, binding agent, disintegrating agent, lubricant etc., make solid preparations such as tablet, granule, powder, powder agent, capsule, liquor commonly used, suspending agent, Emulsion etc.In addition, can not form liquid, and obtain dry product by adding appropriate carriers before it uses yet.

Pharmaceutical carrier can be selected according to above-mentioned dosage form.Oral formulations can utilize starch, lactose, white sugar, mannitol, carboxymethyl cellulose, corn starch, inorganic salt etc.In addition, preparation is during oral formulations, further mixed adhesive, disintegrating agent, surfactant, lubricant, flow promoter, correctives, coloring agent, spice etc.

On the other hand, non-oral formulation, can be according to conventional method, will as effective ingredient of the present invention be selected from the present invention the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, the acid sugar that use pure and mild they salt substance dissolves or be suspended in distilled water for injection as diluent, normal saline, D/W, injection vegetable oil, Oleum sesami, Oleum Arachidis hypogaeae semen, Oleum Glycines, Semen Maydis oil, propylene glycol, the Polyethylene Glycol, add as required biocide, stabilizing agent, etc. preparations such as imbibition reagent, painlessization reagent.

Therapeutic agent of the present invention or preventive can be according to dosage form with suitable route of administration administrations.Medication is had no particular limits, can in use with, external or injection.Injection can also comprise suppository by administrations such as intravenous for example, intramuscular, subcutaneous, Intradermal in the external agent.

Dosage as therapeutic agent of the present invention or preventive, can suitably set according to its dosage form, medication, application target and the patient's age, body weight, symptom etc. that are suitable for this medicine, be variable, contained pure and mild their amount of substance of salt of the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, the acid sugar that use in the present invention 0.01～2000mg/kg every day that preferably is grown up that is selected from the general preparation.Certainly,, lack just enoughly sometimes than above-mentioned dosage, need sometimes above above-mentioned scope because dosage can be according to aforementioned various condition variation.During oral administration, therapeutic agent of the present invention or preventive can be in required administration scope directly former state administration, in addition, also it can be added arbitrarily to daily picked-up in the diet product.Also can produce the raw material use of inducing as somatomedin with being selected from the present invention pure and mild their material of salt of the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, the acid sugar that use with diet product.

Accept the liver of partially hepatectomized, original size is got back in regeneration rapidly.Although the essence for this Hepatopoietin and use thereof is also indeterminate for many years, in acute severe hepatitis patient's blood plasma, found HGF, and from patient's blood plasma, its separation, purification come out (J.Clin.Invest., 88 414-419,1988).Further, cloned people HGFcDNA, and clear and definite the primary structure of HGF (Biochem.Biophys.Res.Commun., 163 967-973,1989).In addition, illustrated the dispersion factor (SF) that promotes cell movement and be same substance (Proc.Natl.Acad.Sci.USA.88 7001-7005,1991 with HGF as the tumor cytotoxicity sex factor (tumor cytotoxic factr (TCF)) of tumor cell obstruction factor; Biochem.Biophys.Res.Commun., 180 1151-1158,1991).

HGF is not only to hepatocyte, and multiple epithelial propagation such as bile duct epithelial cell, renal cells, gastric mucosal cell are had facilitation.In addition, can induce as promoting epithelial mobility or vascularization, the epithelial tube chamber form seen in forming to form, HGF is the multi-functional material that shows colorful physiologically active.That is, HGF induces epithelial propagation promotion when repairing the internal organs obstacle in various internal organs, and forms such as mobility's promotion or vascularization form.

HGF shows the hepatocyte growth effect, and albumen synthesize facilitation, and bile blocks the improvement effect, and the effect of the nephropathy that causes of prophylactic agent etc.From these facts, can expect its therapeutic agent as hepatitis gravis, liver cirrhosis and hepatic bile retardance.But HGF itself does not also reach the degree of practicability as therapeutic agent.Although also tested in the gene treatment and imported the genetic method of HGF, owing to act on the side effect that causes in unwanted time, place, this application is also far from practicability.Like this, can conclude that if HGF can not induce arbitrarily from outside administration, then it is effectively, up to the present, has confirmed IL-1, prostaglandin E in hepatitis, liver cirrhosis, hepatic bile retardance etc. need the treatment of diseases of HGF expression and prevent ₁, E ₂, heparin etc. has inducing action to it.IL-1, prostaglandin E ₁, E ₂HGF is genic to transcribe the generation of inducing HGF by inducing.

On the other hand, known heparin has HGF to produce inducing action, and it is not induced, and HGF is genetic to transcribe, but the generation by promoting that mRNA translation step is afterwards induced HGF.That is, transcribe not derivative state in that HGF is genetic, do not have HGF to produce and induce effect.On the contrary, transcribe in the derivative state in that HGF is genetic, the generation of HGF can be significantly induced in discovery.

In addition, effective ingredient among the present invention not necessarily directly carries out somatomedin such as HGF and transcribes inductive material, but it is to transcribe when being induced for deducibility, can effectively promote to transcribe, the material in the stage after transcribing such as further can promote to translate, its result has the potentiation of the generation of so-called inductive factor.That is, so-called among the present invention " somatomedin generation inducing action " is meant, the enhanced effect that inductive factor produces, and this effect can be by for example using the enhancing of the somatomedin of effective ingredient administration front and back to judge to the people.Here." transcribing when being induced " is meant that for example transcribing when it needs of HGF carried out, by aforementioned effective ingredient, it is transcribed and is further promoted to transcribe initial stage of being promoted at HGF, afterwards, not superfluous generation HGF, therefore, the production of HGF is enhanced when needing in vivo, but induces by the generation that such extremely safe ground carries out HGF.

The inducing material of somatomedin that in therapeutic agent of the present invention or preventive, also can further contain acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt that to work in coordination with increase and use in the present invention.

So-called " can work in coordination with the material of increase " is meant among the present invention, if with effective ingredient of the present invention and this material and usefulness, actively transcribe by this material and to induce, its as a result the somatomedin of effective ingredient of the present invention produce inducing action by the material of collaborative increase.

As the collaborative acidic polysaccharose that uses in the present invention that increases, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or the inducing material of the somatomedin of their salt, increase these acidic polysaccharoses so long as have to work in coordination with, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or the inducing material of the somatomedin of their salt can, this is had no particular limits, for example can enumerate and be selected from cytokine class, prostaglandins, the chemical compound that contains the cyclopentenes ring, the material in minoxidil and the carpronium chloride.In addition, the shogaol that contains in the Rhizoma Zingiberis Recens etc., zingiberol etc., the curcumin that contains in the Rhizoma Curcumae Longae etc. etc. also is to increase the inducing material of HGF, also can be used as the collaborative inducing material of HGF that increases the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt that use in the present invention and uses.

As cytokine class, can enumerate aforementioned IL-1 etc., as prostaglandins, can enumerate aforementioned prostaglandin E ₁, E ₂Deng.

In addition, as chemical compound, can enumerate the chemical compound and the derivant thereof of following formula (III) representative with cyclopentenes ring.

These can use separately, also can mix use more than 2 kinds.

For example, chemical compound with cyclopentenes ring and the prostaglandin E represented respectively of following formula (III)～(V) ₁, E ₂Identical, can induce that HGF is genetic to transcribe, by with the synergism of the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt that use in the present invention, can significantly increase the generation of HGF.Promptly, by being selected from cytokine class, prostaglandins, the chemical compound that contains the cyclopentenes ring, the chemical compound of Rhizoma Zingiberis Recens origin, the chemical compound of Rhizoma Curcumae Longae origin etc. has HGF and transcribes the material of inducing action and be selected from the acidic polysaccharose that uses in the present invention, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or the material in their salt mixes as mixture and usefulness, the acidic polysaccharose that can be used in the present invention, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or the somatomedin that contains of their salt produces inducing action and increased by collaborative, and very high HGF produces and induce effect.

In addition, also this mixture can be produced the raw material of inducing with diet product or feedstuff as somatomedin uses.

For example, the preparation method of the chemical compound of formula (III) representative is recorded in international disclosing in No. 98/13328 communique, the preparation method of the chemical compound of formula (IV) representative is recorded in international disclosing in No. 98/39291 communique, the preparation method of the chemical compound of formula V representative is recorded in international disclosing in No. 98/40346 communique, can prepare these chemical compounds by these preparation methoies.

The preparation method of the chemical compound of formula (III) representative can be any method, can use chemical synthesis synthetic [CarbohydrateRes., the 2478th volume, 217～222 pages (1993), Helvetica Chimica Acta, the 55th volume, 2838～2844 pages (1972)].In addition, can use at least a sugar compounds that is selected from alduronic acid, uronic acid derivative, contains alduronic acid and/or uronic acid derivative, contain alduronic acid and/or uronic acid derivative sugar compounds contain material in the thing, be heated the cyclopentenone or its purification thing that generate in the processing.Chemical compound shown in the formula (IV) for example can obtain by making the reaction of chemical compound shown in the formula (III) and glutathion.In addition, chemical compound shown in the formula V can obtain by making the reaction of chemical compound shown in the formula (III) and propionic andydride.

In therapeutic agent of the present invention or preventive, the amount of the inducing material of somatomedin of the acidic polysaccharose that collaborative increase is used in the present invention, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt is so long as can to work in coordination with the degree that increases this inducing action just passable, this is had no particular limits, be generally the amount of the preferred 0.001～2000mg/kg of per day for adults.The collaborative material that increases this inducing action can merge with the material that is selected from the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt that use in the present invention carry out preparationization, also can carry out preparationization respectively.Therefore the method for preparationization, administering mode can carry out according to the method for putting down in writing in this description, and can obtain the desired somatomedin of the present invention and produce and induce by the collaborative effect that increases.

It is active that the acidic polysaccharose of Shi Yonging, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt also have heparitinase (ヘ パ ラ Na one ゼ) inhibition in the present invention, and cancerometastasis suppresses active, angiogenesis inhibiting activity.Therefore, can make, provide cancer transfer inhibitor, angiogenesis inhibitor with the material that is selected from these materials as effective ingredient.Particularly chemical compound has very strong heparan enzyme inhibition and cancerometastasis inhibitory action shown in the formula of fucoidan origin (I), and containing this chemical compound is exceedingly useful as the pharmaceutical composition of effective ingredient as cancer transfer inhibitor.In addition, the diet product that contain this compound formation are very valuable with, inhibition angiogenesis with the diet product as suppressing cancerometastasis.

Contain that to have a somatomedin inducing, be selected from the acidic polysaccharose that uses in the present invention, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or the somatomedin that forms of the material of their salt produces to induce and uses food, beverage or feed link are crossed its somatomedin and are produced inducing action, are improving, the acidic polysaccharose of prevention to using in the present invention, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or their salt show susceptibility to need somatomedin to produce in the physical qualification of the symptom of inductive disease or improvement biology described later be exceedingly useful.

In addition, food of the present invention, beverage or feedstuff, or said in the aftermentioned cosmetics " containing " comprise the meaning that contains, adds, dilutes.Contain the state that contains effective ingredient of the present invention in food, beverage or the feedstuff that is meant, interpolation is to point to the state that adds effective ingredient of the present invention in the raw material of food, beverage or feedstuff, and dilution is meant the feed states of adding food, beverage or feedstuff in the effective ingredient that the present invention uses.

In addition, further contain the collaborative inducing aforementioned substances of somatomedin that increases, for example, be selected from cytokine class, prostaglandins, have the material of the chemical compound of cyclopentenes ring, consider it is preferred from the viewpoint that helps to improve, prevent aforementioned diseases or improve physical qualification.

In addition, in diet product of the present invention, aforementioned effective ingredient, somatomedin, maybe can to work in coordination with the preferred condition that increases the inducing material of somatomedin identical with the situation of aforementioned therapies agent or preventive.Particularly as diet product of the present invention or feedstuff, from improving hepatopathy, improve nervous system disease, improving the viewpoint of diabetes, preferably hepatocyte proliferation factor produces and induces usefulness, Insulin-Like multiplicaiton factor to produce to induce usefulness or nerve growth factor is long-living induces with diet product or feedstuff.

The manufacture method of Foods or drinks of the present invention has no particular limits this so long as can obtain having inducing this Foods or drinks of somatomedin and get final product.For example, cooperation, conditioning, processing etc. can be according to the methods of using in the normal food, preparation method by these Foods or drinkses can prepare Foods or drinks of the present invention, can contain in the prepared Foods or drinks to have the inducing material of the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt that use in the present invention that is selected from of somatomedin as effective ingredient.

Foods or drinks of the present invention is had no particular limits, can enumerate such as grain processed goods (wheat flour processed goods, the starch based processed goods, the premixing processed goods, such as noodles, alimentary pastes, Bread and Pastries, the filling class, the Semen Fagopyri Esculenti Noodles, bran, rice flour, bean noodles, packing cake etc.), oils and fats processed goods (plastic fat, fried food oil, salad oil, the mayonnaise class, flavoring agent etc.), soybean processing product (bean curd, bean milk, fermented soybean etc.), meat packing product (Petaso, bacon, the compacting Petaso, sausage etc.), aquatic product (the freezing ground flesh of fish, breaded fish stick, the grilled fish volume, the square cake of flesh of fish Rhizoma Dioscoreae, a fish fry ball, quick-boil fish meatball, muscle, fish ham, sausage, wooden fish, the roe processed goods, canned aquatic products, the fish of boiling with billot, the pickles of shellfish one class etc.), milk product (raw material milk, cheese, Yoghurt, butter, cheese, condensed milk, the powder breast, ice river in Henan Province icepro etc.), vegetable, the fruit processed goods (is stuck with paste class, the beans class, pickle the thing class, fruit drink, vegetable beverage, bland etc.), confection class (chocolate, Biscuits, band filling bread, cake, rice flour dessert, rice cake class etc.), alcoholic beverage (Janpanese sake, Chinese wine, wine, Whiskey, Japan's liquor, Little water., brandy, gin, rum, medicated beer, refrigerant alcoholic beverage, wine, liqueur etc.), hobby beverage (green tea, black tea, oolong tea, coffee, refreshment drink, lactic acid beverage etc.), flavouring agent (soy sauce, sauce, vinegar, sweet rice wine etc.), canned bottle pack dress food (beef rice, the still meal, RED BEAN RICE, the curry meal, other various conditioned food), half-dried or reserve ration (pork liver paste, tablespread, the juice of Fagopyrum esculentum Moench noodles, the condensed soup class), dried foods (instant noodles, instant curry meal, instant coffee, juice powder, powder, the instant dip, conditioned food, conditioned beverage, conditioned soup etc.), frozen food (sukiyaki, egg custard, Broiled River Eel, hamburger minced beef cutlet, steamed dumplings, dumpling, various ice lollies, fruit cocktail etc.), solid food, liquid food (soup etc.), farming and forestry processed goods such as spice, the animal husbandry processed goods, aquatic products processing product etc.

As Foods or drinks of the present invention, contain and be selected from material with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt, as long as contain the required amount of its physiological function of expressing, its shape is had no particular limits, comprise the ingestible shapes of oral administration such as drug sheet, graininess, capsule.In addition, sulfated polysaccharides and degradation product thereof with the inducing algae origin of somatomedin, for example fucoidan and degradation product thereof are exceedingly useful as the raw material of the health food that has this physiological action and dietary fiber function simultaneously, the manufacturing raw material of Foods or drinks.

Have no particular limits for being selected from the content of material (effective ingredient) in Foods or drinks in the Foods or drinks of the present invention with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt, can suitably select from function and physiologically active, but content of effective, for example, per 100 weight portion food contain 10 ^-9More than the weight portion, preferred 10 ^-7～2 weight portions, for example, per 100 weight portion beverages contain 10 ^-9More than the weight portion, preferred 10 ^-7～2 weight portions.

In addition, per day for adults is preferably absorbed effective ingredient and is reached 0.01～2000mg/kg, can reach the desirable oral administration of the present invention like this and carry out somatomedin and produce inductive effect.

In addition, the present invention also provides and has contained the biology feedstuff that is selected from the material formation with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt.

And biological method for breeding further is provided, it is characterized in that using this forage feed biology.

In addition, provide biological raising agent, it is characterized in that containing and be selected from material with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt.

In the present invention, biology is meant for example cultivated animals, pet animals etc., can enumerate domestic animal, experimental animal, poultry, Fish, Crustaceans or shell-fish as cultivated animals.

Can enumerate based on somatomedin is inducing as feedstuff and to improve the feedstuff that health is used.

Raise agent as biology and can enumerate impregnating agent, feed additive, beverage additive.

In the present invention, be selected from material and have biological feeding efficiency, for example effects such as survival rate, fattening rate, spawning rate, product rate, wean rate of raising with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt.

Be selected from material with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt, usually give and 0.01～2000mg every day for object organisms 1kg body weight, can in the raw material of artificial mixed fodder, mix and add, also can add to again to mix in other raw material and add with after the powder stock of artificial mixed fodder mixes.

Have no particular limits with the content in the feedstuff in the object organisms that finally obtains for being selected from material with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt, can suitably use according to purpose, be suitable in the ratio of 0.001～15 weight %.For example, when being purpose to improve liver function, the ratio of 0.01～10 weight % is suitable.

As artificial mixed fodder, can enumerate animal raw materials such as fish flour, casein, squid meat, vegetable raw materials such as soybean meal, wheat flour, starch, feedstuff are with microorganism raw materials such as yeast, animal raw fats such as cod liver oil, squid liver oil, soybean oil, Semen Allii Tuberosi wet goods vegetative grease, vitamins, minerals, amino acids, antioxidant etc. are as the artificial mixed fodder of raw material.In addition, can enumerate Fish such as oppressing mince and use feedstuff.

Preparation method for feedstuff of the present invention has no particular limits, in addition, can be according to general feedstuff mixed preparing, as long as contain the material that being selected from of effective dose has the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt in the feedstuff of manufacturing.

In addition, be selected from water that the material with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt can directly add pond, tank, water reservoir or the field of raising to, the sea water etc., or by object organisms is flooded to.This dipping method is effective especially when the feedstuff intake of object organisms reduces.

The concentration that has the material of the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt for being selected from water or the sea water has no particular limits, can suitably use according to purpose, the ratio of preferred 0.00001～1 weight % is suitable.

In addition, also can absorb by object organisms with beverage as raising containing the beverage that is selected from material with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt.

The concentration that has the material of the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt for being selected from this beverage has no particular limits, can suitably use according to purpose, the ratio of preferred 0.0001～1 weight % is suitable.

Contain and be selected from material and raise agent as the biology that effective ingredient forms with the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt, for example impregnating agent, feed additive, beverage additive can be respectively with known mixing and manufacture method making.

As the biology that is suitable in the present invention, have no particular limits, can enumerate horse as aquaculture organism, cattle, pig, sheep, goat, camel, domestic animals such as Luo Yang, mice, rat, guinea pig, experimental animals such as rabbit, chicken, duck, turkey, poultry such as Ostriches, snapper, oplegnathus fasciatus, Paralichthys olivaceus, the butterfly fish, the Yellowtail fish, ocyurus chrysurus, yellow bar Yellowtail, tuna Da Jia Ajigasawa, fragrant fish, the salmon class, red fin circle Puffer, anguilla japonica, Misgurni anguillicaudati, Fish such as cat fish, prawn, black Tiger Prawns (プ ラ ッ Network ィ ガ one), prawn ( ィ シ ョ ゥ ェ PVC), shell-fish such as mud crab etc., Bao, beautiful mouthful of spiral shell belongs to, scallop, shellfishes such as Concha Ostreae, can enumerate Canis familiaris L. as pet animals, cats etc. can be widely used in the animal in the terrestrial water.

Can contain to be selected from by picked-up and have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or the material of their salt, or object organisms be impregnated in to contain to be selected from have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, or the liquid of the material of their salt, improve domestic animal, experimental animal, poultry, Fish, Crustaceans, shellfish, healths such as pet animals, its result, can prevent or the microbism of treatment target biology, viral infectious disease, and can significantly improve the disease symptoms of infection animal.In addition, the health of object organisms be can keep, its survival rate, rate of growth, spawning rate, product rate, wean rate, fertility rate etc. significantly improved.

In addition, these cultivated animals exist (1) that the disease that bacterial infection causes often takes place, owing in restricted portion, culture, in case generation infectious disease, very fast infected whole death that cause, (2) be easy to take place parasitic disease, nutritional disease, environmental disease, tumor etc., (3) domesticated animal pressure is big in narrow region, body surface can take place raised the abrasive situation of facility, and individuality is easy to be adhered to by antibacterial or parasite, (4) in addition, because problems such as pressure, food intake are seldom, and it is slow to grow up, but feedstuff of the present invention, improve the effect of health by it, can be reduced in the pressure of domesticated animal in the narrow zone significantly, do not raise the scratch of facility body surface, make honey stomach, can significantly improve rate of growth, produce sub-rate, spawning rate, the wean rate, disease prevention rate etc.

Having the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt is useful as the effective ingredient of cosmetics, according to the present invention, can provide somatomedin with the material that is selected from the acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt that use in the present invention as effective ingredient, for example HGF produces to induce and applies some make up.

In addition, contain the collaborative inducing aforementioned substances of somatomedin that increases, for example, be selected from cytokine class, prostaglandins contains the material of the chemical compound of cyclopentenes ring, sees it is preferred from the viewpoint that reaches desirable effect.

In addition, in cosmetics of the present invention, the preferred condition of aforementioned effective ingredient, somatomedin or the inducing material of collaborative increase somatomedin is identical with aforementioned therapies agent or preventive.Particularly, as cosmetics of the present invention, from activating epithelial viewpoint, preferably hepatocyte proliferation factor produces and induces usefulness, the generation of the Insulin-Like factor to induce usefulness or nerve growth factor production to induce the cosmetics of usefulness.

Effective ingredient as these cosmetics, preferred especially fucoidan and degradation product thereof, can provide for example F-fucoidan and/or its degradation product, or the chemical compound shown in the formula (I) produces and induce usefulness as the somatomedin that has that effective ingredient forms, for example HGF produces to induce and uses biological cosmetics.Induce the content of acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt in applying some make up to be generally 0.001～20 weight % in the somatomedin generation, more preferably 0.001～5 weight %.

Somatomedin of the present invention produces to induce and for example uses, HGF produces to induce and applies some make up, can make according to known mixing conventional method, produce to induce as somatomedin of the present invention and apply some make up, comprise for example lotion class, emulsion class, cream kind, facial film class, bathe and use class, the class of washing one's face is bathed with soap or bath with detergent etc.

Cosmetics of the present invention according to the employed amount of its purposes form can be respectively, for example during lotion, and situation about using for example for the whole face of people, preferred 0.01～the 5g of each use, more preferably about 0.1～2g,, therefore can obtain the so-called skin Caring effect of the present invention owing to can activate epithelial cell.

The present invention also provides and has contained the material that is selected from acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt and produce derivant as the somatomedin of effective ingredient, this generations derivant the functional study of somatomedin, and the somatomedin relevant disease be useful with the screening of medicine.

In addition, the present invention also provides and has contained the material that is selected from acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt and produce regulator as the somatomedin of effective ingredient.

Somatomedin of the present invention produces derivant, somatomedin produces regulator and uses above-mentioned effective ingredient, and available known preparation legal system prepares agent.Produce derivant as somatomedin, can enumerate above-mentioned therapeutic agent etc. as an one example.In addition, somatomedin of the present invention produces regulator and is meant in vivo, transcribes the preparation that the inductive initial stage promotes that somatomedin is transcribed at somatomedin.Produce regulator by somatomedin of the present invention and have the generation that only strengthens somatomedin, and can not make the remarkable result of the generation surplus of somatomedin at the state that needs somatomedin to produce.

The fucoidan of Shi Yonging and/or its degradation product have strong especially somatomedin generation inducing action in the present invention, somatomedin produces regulating action, is exceedingly useful as the effective ingredient that uses in preparation of the present invention.

The inducing heparin of the known in the past HGF of having does not promote the transcribing of mRNA of HGF, but fucoidan and degradation product thereof are transcribed the initial stage that is promoted at the mRNA of HGF, further promote transcribing of its mRNA.In vivo, usually do not carry out the transcribing of mRNA of HGF, just transcribe where necessary.Fucoidan and degradation product thereof, 7～12SFd-F for example described later, only the initial stage that is promoted of transcribing of HGF further promotes it to transcribe in vivo, can superfluously not produce HGF afterwards, the production status of HGF has only when needing in vivo just and is promoted, from this point, be that the HGF of extremely safe produces the adjusting material.

Therefore, in other scheme of the present invention, aforementioned therapies agent or preventive, diet product etc. can be directly used in the adjusting somatomedin and produce inductive purpose.Produce the dosage of regulator as somatomedin, as long as the generation of scalable somatomedin, this is had no particular limits, but the dosage of this effective ingredient in the present invention, the dosage that for example can enumerate for people's effective ingredient is preferred 0.01～2000mg/kg (body weight).

In addition, use use in the present invention have an inducing acidic polysaccharose of somatomedin, even for example fucoidan and/or its degradation product for rat with the oral single-dose of 1g/kg, also find dead example.In addition, dextran sulfate sodium also is safe chemical compound.In addition, other acidic polysaccharose of Shi Yonging, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt to the rat oral administration, are not all found toxicity with its physiology effective dose in the present invention.

In addition, as another program, need somatomedin to produce inductive treatment of diseases agent or preventive the invention provides, it is that the inducing extract of somatomedin that has that will be selected from Radix Artemisia ordosicae extract, Fructus Momordicae charantiae extract, aloe extract, Caulis et Folium Chrysanthemi segeti extract, Chlorella extract and Spirullina extract forms as effective ingredient.

In addition, provide and contain the inducing extract of somatomedin that has that is selected from Radix Artemisia ordosicae extract, Fructus Momordicae charantiae extract, aloe extract, Caulis et Folium Chrysanthemi segeti extract, Chlorella extract and Spirullina extract and produce as the somatomedin of effective ingredient and induce with diet product or feedstuff.

In addition, provide and contain the inducing extract of somatomedin that has that is selected from Radix Artemisia ordosicae extract, Fructus Momordicae charantiae extract, aloe extract, Caulis et Folium Chrysanthemi segeti extract, Chlorella extract and Spirullina extract and produce to induce as the somatomedin of effective ingredient and apply some make up.

The purification of the aforementioned extract that extracts from these plants, microorganism can carry out with following known method.Gather fruit as material plant, seed, leaf, stem, root, rhizome etc. in suitable period, or microorganism, directly or after carrying out drying steps such as air drying, form extraction feed.When raw material is the squeezeding juice of plant or resin, can directly use as extraction feed.

Extracting the extract that contains aforementioned effective ingredient from above-mentioned dry animal and plant body, microorganism can carry out according to following known method.With raw material pulverizing or after chopping up, use batch type or continous way extracting process to carry out with solvent.As extractant, can make ketones such as alcohols such as water, chloroform or ethanol, methanol, isopropyl alcohol, acetone, butanone, hydrophilic or oil loving solvents such as methyl acetate, ethyl acetate can use separately or use with mixed liquor.Extraction temperature is generally 0～150 ℃, preferably carries out at 5～120 ℃.

When carrying out with batch type, the extraction time is about 10 minutes～20 days, and the usual solvents use amount is equivalent to 1～30 times of weight of raw material, preferred 2～20 times of weight.Extracting operation can be to flood placement by stirring or passing through, or is used in combination.Extracting operation can be as required 2～3 times repeatedly.Can enumerate the method for the soxhlet apparatus that use is combined to form reflux cooler and siphon as the continuous extraction method, quantity of solvent, extraction time etc. are identical with the condition of aforementioned batch type extracting process.

Contain in the extract that the present invention uses in the thick extract that obtains in the operation early stage by the residue that filters or centrifugalize is removed.Insoluble sludge is also had by the occasion of using as active residue in addition.

The purification active component can use known purification process from the plant extract active component from thick extract, preferably uses two-phase solvent partition method, column chromatography etc., can be used singly or in combination.

The gained extract as effective ingredient, can be made medicine, diet product, feedstuff and cosmetics etc. according to purpose.Its manufacture method can be carried out according to the preceding method described in the 1st～3rd invention of the present invention.

Can produce inducing action according to its somatomedin according to the amount of extract in each purpose goods and determine, in common goods, preferred 0.001～100 weight %, more preferably 0.01～30 weight, further preferred 0.1～20 weight %.

In addition, these extracts that use among the present invention to the rat administration, are not found toxicity with its effective dose.

Below, enumerate embodiment, the present invention is described more specifically, but the present invention is not limited to these embodiment.In addition, the % in the mixing of each composition is weight % among the embodiment.Reference example 1

(1) with after the ガ go メ Thallus Laminariae (Thallus Eckloniae) intensive drying, dry thing 20kg pulverizes with Jiyu mill (nara machinery is made made).

In 900 liters in tap water, dissolve calcium chloride dihydrate (Japanese Cao Da society system) 7.3kg, mix ガ go メ Thallus Laminariae (Thallus Eckloniae) ground product 20kg then.Make the liquid temperature be warming up to 90 ℃ of heating 40 minutes by 12 ℃ by being blown into steam, under agitation 90～95 ℃ of insulations 1 hour, cooling obtains 1100 liters of refrigerants then.

With equipment for separating liquid from solid (the system CNA of West Farrier Separator society type) refrigerant is carried out solid-liquid separation, prepare about 900 liters solid-liquid separation supernatant.

360 liters of FE10-FC-FUS0382 with DAICEL society system of solid-liquid separation supernatant (classification molecular weight 30,000) are concentrated into 20 liters.Add 20 liters in tap water, and then be concentrated into 20 liters, repeatedly should operation 5 times, carry out desalting processing, obtain 25 liters of the extracting solution of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin.

This extracting solution is carried out lyophilization for 1 liter, obtain the dry thing 13g of fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin.

According to the method described above from the mince dry thing of fucoidan of preparation Thallus Laminariae (Thallus Eckloniae) origin of Thallus Laminariae (Thallus Eckloniae) xeraphium.And the dry thing of fucoidan for preparing equally, the Macrocystis origin from the dried powder (sale of trade name Seaweed Powder:Andesu trade Co., Ltd.) of Macrocystis (Lessonia nigrescence).

(2) the dry thing 7g of the fucoidan that reference example 1-(1) is put down in writing is dissolved in and contains among 50mM sodium chloride and 10% alcoholic acid 20mM imidazole buffer (pH8.0) 700ml, removes insoluble matter by centrifugalize.With DEAE-Cellulofine A-800 post (φ ll.4cm * 48cm) with identical buffer equilibrating, supernatant after the centrifugalize is added on the pillar, with the same buffer washing, with 50mM sodium chloride eluting (1 fraction: 250ml) to the 1.95M Concentraton gradient.With phenol sulfuric acid process and carbazole sulfuric acid process, obtain total sugar amount and glucuronic acid content, eluting obtains fraction 43～49 successively, fraction 50～55, the component of fraction 56～67.Then, these components by electrodialysis desalination postlyophilization, are prepared component I (340mg) from fraction 43～49 respectively, prepare component I I (870mg), prepare component III (2.64g) from fraction 56～67 from fraction 50～55.

The DEAE-Cellulofine A-800 post elution curve that in Fig. 1, shows the fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin.In Fig. 1, the longitudinal axis is with the carbazole sulfuric acid process absorbance (stain among the figure) at 530nm, and (mS/cm: white square among the figure), transverse axis is the fraction sequence number in the absorbance (white point among the figure) of 480nm and electrical conductivity with the phenol sulfuric acid process.Reference example 2

(1) will replace zygosaccharomyces SN-1009 (FERM BP-5747) is inoculated in to have injected and contains glucose 0.25%, peptone 1.0%, (120 ℃ of culture medium 600ml that the artificial seawater of yeast extract 0.05% (JamarinLaboratory society system) pH8.2 forms and sterilizations, 20 minutes) 2 liters of conical flasks in, cultivate 26 hours as planting culture fluid at 25 ℃.To contain peptone 1.0%, yeast extract 0.02%, culture medium 20 liftings that the artificial seawater pH8.0 of the sulfated polysaccharides 0.2% of record and defoamer among the following reference example 2-(2) (the system KM70 of chemical industry society of SHIN-ETSU HANTOTAI) 0.01% forms were gone in the jar fermenter of 30 liters of volumes, 120 ℃ of sterilizations 20 minutes.After the cooling,,, cultivated 24 hours under the mixing speed that ventilation that per minute is 10 liters and per minute 250 change at 24 ℃ with above-mentioned kind of culture fluid 600ml inoculation.After cultivation is finished, medium centrifugal is separated, obtain thalline and culture supernatant.After the gained culture supernatant concentrated by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 10,000, saturated ammonium sulfate with 85% is saltoutd, collect the precipitation that generates by centrifugalize, 20mM Tris-hydrochloride buffer (pH8.2) with the artificial seawater that contains 1/10 concentration is fully dialysed, and preparation 600ml has the tip type sulfated polysaccharides digestive enzyme liquid of selectively acting to sulfated polysaccharides.

(2) exsiccant ガ go メ Thallus Laminariae (Thallus Eckloniae) 2kg is pulverized with the cutting grinding machine (increasing good fortune industry society system) of the filter screen that is equipped with diameter 1mm, gained Thallus Laminariae (Thallus Eckloniae) sheet is suspended in 20 liter of 80% ethanol, stirred 3 hours at 25 ℃, behind filter paper filtering, that residue is fully clean.Residue obtained 40 liters of being heated to 95 ℃ of being suspended in are contained among the 20mM sodium phosphate buffer pH6.5 of 50mM sodium chloride, handled 2 hours at 95 ℃ when intermittently stirring, extract sulfated polysaccharides.

Float in the extract is filtered, and after the preparation filtrate, filtration residue is cleaned with 3.5 liters of 100mM sodium chloride, further obtains filtrate.

After the merging of two filtrates, be cooled to 30 ℃, the alginate lyase (the biochemical industrial society of Nagase system) of interpolation 3000U adds 4 liters of ethanol afterwards, stirs 24 hours at 25 ℃.Then, carry out centrifugalize, the gained supernatant further no longer is filtered off with containing 10% alcoholic acid 100mM sodium chloride continuous ultrafiltration to coloring material after being concentrated into 4 liters by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000.

The precipitation that generates in the non-filtrate is removed by centrifugalize, and this supernatant is cooled to 5 ℃, transfer to pH2.0 with 0.5N hydrochloric acid after, remove the precipitations such as protein of generation with centrifugalize, the gained supernatant transfers to pH8.0 with the 1N sodium hydroxide rapidly.

Then, carry out ultrafiltration, after the complete replacement solvent of 20mM sodium chloride pH8.0, transfer to pH8.0 once more, after the centrifugalize, carry out lyophilization, obtain the sulfated polysaccharides of about 95g by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000.

(3) exsiccant ガ go メ Thallus Laminariae (Thallus Eckloniae) 2kg is pulverized with the cutting grinding machine of the filter screen that is equipped with diameter 1mm, gained Thallus Laminariae (Thallus Eckloniae) sheet is suspended in 20 liter of 80% ethanol, stirred 3 hours at 25 ℃, behind filter paper filtering, that residue is fully clean.In the residue obtained 20 liters of buffer (pH8.2) that are suspended in the tip type sulfated polysaccharides digestive enzyme liquid 30ml, 10% ethanol, 100mM sodium chloride, 50mM calcium chloride and the 50mM imidazoles that contain above-mentioned reference example 2-(1) preparation, stirred 48 hours at 25 ℃.With the stainless steel metal silk screen filter of this suspension with mesh diameter 32 μ m, residue is cleaned with 10% ethanol that contains 50mM calcium chloride.Again residue is suspended in 10 liters of 10% ethanol that contain 50mM calcium chloride, stirs after 3 hours, use the stainless steel metal silk screen filter, clean.Further with residue behind the similarity condition low suspension, stirred 16 hours, with the stainless steel metal silk screen filter of diameter 32 μ m, clean.

Collect the filtrate and the cleaning mixture that so obtain,, be separated into filtrate and non-filtrate by being equipped with the ultrafilter ultrafiltration of the hollow fibre of getting rid of molecular weight 3000.

After this filtrate was concentrated into about 3 liters with rotary evaporator, centrifugalize obtained supernatant.The gained supernatant adds calcium acetate and makes it to reach 0.1M by being equipped with the electrodialyzer desalination of the film of getting rid of molecular weight 300 in this solution, remove the precipitation of generation by centrifugalize.Supernatant is added on DEAE-Cellulofine (4 liters of the amount of resin) post of the calcium acetate equilibrating of using 50mM in advance, after fully cleaning with 50mM calcium acetate and 50mM sodium chloride, with the sodium chloride gradient elution of 50mM～800mM.Carry out with every bottle of 500ml of fractional distillation amount this moment.When analyzing the fractional distillation component with cellulose acetate membrane electrophoresis method [Analytical Biochemistry the 37th volume, 197～202 pages (1970)], near the sulfated polysaccharides (fraction 63) that eluting goes out during the about 0.4M of sodium chloride concentration is a homogeneous.

Then, at first with the liquid concentration of fraction 63 to 150ml, add sodium chloride and make concentration reach 4M, it is added on phenyl-Cellulofine (amount of resin 200ml) post of the sodium chloride equilibrating of using 4M in advance, fully clean with the sodium chloride of 4M.Collect the sulfated sugar component of non-adsorptivity,, obtain desalinization liquor 505ml by being equipped with the electrodialyzer desalination of the film of getting rid of molecular weight 300.

40ml in the gained desalinization liquor is added to contains the equilibrated Cellulofine GCL-900 of 10% alcoholic acid 0.2M sodium chloride and (on the post of 4.1cm * 87cm), carry out gel filtration.Collect with each fraction 9.2ml.

With phenol sulfuric acid process [Analytical Chemistry the 28th volume, 350 pages (1956)] whole fractions are carried out the total sugar component analysis.

As a result, because sulfated sugar forms a peak, collect the middle body fraction 63～70 at this peak, after the electrodialyzer desalination that is equipped with the film of getting rid of molecular weight 300, lyophilization obtains the dry product of chemical compound shown in the 112mg following formula (VI).Below, this chemical compound is called 7-12SFd-F.

(4) Tris-hydrochloride buffer (pH7.6) 16ml, the 1M CaCl of adding 1M in 2.5% aqueous solution 80ml of the III component (F-fucoidan) that reference example 1-(2) prepares ₂Tip type sulfated polysaccharides digestive enzyme liquid 8ml, distilled water 176ml that aqueous solution 16ml, 4M NaCl aqueous solution 24ml, reference example 2-(1) obtain were 30 ℃ of heating 3 hours.The F-fucoidan solution that this enzyme is handled is 2% with the ultimate density that rotary evaporator is concentrated into the F-fucoidan, and the operation of dialysing in distilled water then prepares the F-fucoidan aqueous solution that 2% enzyme is handled.With this test material with HPLC (post: SB802.5, column temperature: 35 ℃, mobile phase: 50mM NaCl, flow velocity L0.5ml/min detects; RI ATT=8) analyzes.As a result, have an appointment in this test material 40% 7-12SFd-F.Reference example 3

(1) exsiccant ガ go メ Thallus Laminariae (Thallus Eckloniae) 2kg is pulverized with the cutting grinding machine (increasing good fortune industry society system) of the filter screen that is equipped with aperture 1mm, gained Thallus Laminariae (Thallus Eckloniae) sheet is suspended in 20 liter of 80% ethanol, stirred 3 hours, filter afterwash at 25 ℃.Be suspended in the 20 liters of 30mM imidazole buffers (pH8.2) that replace zygosaccharomyces SN-1009 (FERM BP-5747) tip type sulfated polysaccharides digestive enzyme 1U that contain 50mM calcium chloride, 100mM sodium chloride, 10% ethanol and above-mentioned reference example 2-(1) preparation residue obtained, stirred 2 days at 25 ℃.Then, with the stainless steel metal silk screen filter of aperture 32 μ m, clean.Be suspended in the 40 liters of sodium phosphate buffers (pH6.6) that contain 100mM sodium chloride, 10% ethanol and 4g alginate lyase (the biochemical industrial society of Nagase system) residue obtained, after 4 days, centrifugalize obtains supernatant 25 ℃ of stirrings.For removing the alginic acid low molecular weight product that contains in the gained supernatant, be concentrated into 2 liters by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000 after, carry out solution and exchange with containing 10% alcoholic acid 100mM sodium chloride.After the 400mM calcium acetate that adds equivalent in this solution stirs, centrifugalize, the gained supernatant transfers to pH2 with 1N hydrochloric acid with ice-cooled the time.The precipitation of generation is removed in centrifugalize, and the gained supernatant transfers to pH8.0 with the sodium hydroxide of 1N.With behind this solution ultrafiltration and concentration to 1 liter, carry out the solution exchange with 100mM sodium chloride.Remove the precipitation that generate this moment by centrifugalize.For removing the lyophobic dust in the gained supernatant, in supernatant, add sodium chloride and make it to reach 1M, it is added to the equilibrated 3 liters of phenyl Cellulofine posts of 1M sodium chloride (biochemical industry system), collect effusive component.After this component concentrated with ultrafilter, carry out solution with the sodium chloride of 20mM and exchange lyophilization.The weight of lyophilization thing is 29.3g.

(2) above-mentioned lyophilization thing 15g is dissolved in contains 400mM sodium chloride and cultivate the international Flavobacterium SA-0082 (FERM BP-5402) that puts down in writing in No. 97/26896 description that discloses, 1.5 liters of 50mMTris-hydrochloride buffers of the tip type sulfated polysaccharides digestive enzyme 9U that obtains from this culture, after 6 days, be concentrated into about 300ml 25 ℃ of reactions with vaporizer.Concentrated solution is put into the Dialysis tubing of getting rid of molecular weight 3500 thoroughly dialyses, liquid remaining in the Dialysis tubing is added in 4 liters of DEAE-Cellulofine A-800 posts using the equilibrating of 50mM sodium chloride, after fully cleaning with 50mM sodium chloride, carry out the Concentraton gradient eluting with the sodium chloride of 50～650mM.Further with the sodium chloride abundant eluting of this post with 650mM.Component with the sodium chloride eluting of 650mM in the component that eluting is gone out is collected as the sulfated fucogalactan component, after concentrating with the ultrafilter of getting rid of molecular weight 100,000, sodium chloride with 10mM carries out the solution displacement, lyophilization obtains the lyophilization thing 0.85g of sulfated fucogalactan.The sugar formation of gained sulfated fucogalactan contains galactose and fucose, and its mol ratio is about 2: 1.Reference example 4

The sulfated polysaccharides 120g of reference example 2-(2) preparation is suspended in 8 liters of 20mM imidazole buffers (PH7.5) of the tip type sulfated polysaccharides digestive enzyme that contains 20mM calcium chloride, 300mM sodium chloride, 10% ethanol and 10U reference example 2-(1) preparation, stirred 3 days at 25 ℃, use is equipped with the ultrafilter of the hollow fibre of getting rid of molecular weight 100,000, carries out ultrafiltration when adding above-mentioned buffer.

The tip type sulfated polysaccharides digestive enzyme that adds 34U reference example 3-(2) preparation in ultrafiltrate stirred 2 days at 25 ℃, carried out ultrafiltration by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000, carried out ultrafiltration when adding water.

Collect filtrate, after being concentrated into 1.5 liters with vaporizer, with the complete desalination of desalter, join on 3 liters of DEAE-Cellulofine A-800 posts using 5mM imidazoles-hydrochloride buffer (PH6.5) equilibrating that contains 30mM sodium chloride in advance, after cleaning with 6 liters of same buffer, carry out eluting by Concentraton gradient with the sodium chloride of 30mM～500mM.The required liquid measure of eluting is 48 liters.Eluent is got by every bottle of 180ml branch, measured its sugar content by phenol～sulfuric acid process.Be determined at the absorbance of 232nm simultaneously.Because the component that goes out with the sodium chloride eluting of 130mM～170mM forms a peak, therefore, collects this component, after the desalination of usefulness desalter, lyophilization obtains the oligosaccharide of 5.85g.The quality analysis of this oligosaccharide shows that molecular weight is 1128, and it is the chemical compound shown in the following formula (VII) for the NMR analysis confirmation.Below, this chemical compound is called 6-2S.

Reference example 6

After the cutting grinding machine fragmentation of the dry thing 1kg of commercially available Thallus Laminariae with the filter screen that is equipped with aperture 1mm, be suspended in 10 liter 80% the ethanol, stir after 3 hours, use filter paper filtering, obtain residue.Residue is suspended in 20 liters of the 40mM phosphate buffers (PH6.5) that contain 50mM sodium chloride, handled 2 hours at 95 ℃.After treatment fluid is cooled to 37 ℃, adds ethanol and make it to reach 10%, add commercially available alginate lyase K (the biochemical industrial society of Nagase system) 12000U after, stirring at room 24 hours.With the centrifugalize of gained treatment fluid, supernatant is concentrated into 2 liters by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000 after, remove the precipitation of generation by centrifugalize.After the gained supernatant was cooled to 5 ℃, the hydrochloric acid that adds 0.5N transferred to PH2.0, stirs afterwards 30 minutes, removes the precipitation of generation by centrifugalize.With the sodium hydroxide of 0.5N the PH of supernatant is transferred to 8.0, solution is replaced into the sodium chloride of 20mM by ultrafiltration.After the PH of solution transferred to 8.0, centrifugalize with the lyophilization of gained supernatant, obtained the fucoidan of 90.5g Thallus Laminariae origin.Reference example 7

The dry thing 1kg of the Fucus Vesiculosus (Fucus vesiculousus) of pulverizing is suspended in 10 liter of 80% ethanol, stirs after 3 hours, use filter paper filtering, obtain residue.Residue is suspended in 30 liters of the 30mM phosphate buffers (PH6.0) that contain 100mM sodium chloride, handled 2 hours at 95 ℃.After treatment fluid is cooled to 37 ℃, adds the 100g active carbon and stirred 30 minutes.After adding commercially available alginate lyase K 3000U, add ethanol and make it to reach 10%, stirring at room 24 hours.With the centrifugalize of gained treatment fluid, supernatant is concentrated into 2 liters by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000 after, remove the precipitation of generation by centrifugalize.Carry out ultrafiltration when in this supernatant, adding extract, remove pigment.After the non-filtrate of gained was cooled to 5 ℃, the hydrochloric acid that adds 0.5N transferred to PH2.0, stirs afterwards 30 minutes, removes the precipitation of generation by centrifugalize.With the sodium hydroxide of 0.5N the PH of supernatant is transferred to 8.0, solution is replaced into the sodium chloride of 20mM by ultrafiltration.After the PH of solution transferred to 8.0, centrifugalize with the lyophilization of gained supernatant, obtained the fucoidan of 71g Fucus Vesiculosus origin.

According to the method described above, the fucoidan for preparing the Ascophyllum nodosum origin from the dry thing powder (sale of trade name Algin Gold:Andesu trade Co., Ltd.) of Ascophyllum nodosum (Ascophyllum nodosum).Reference example 8

To be dissolved in the 100ml water according to the fucoidan 2g of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of the method preparation of reference example 1-(1) record, transfer to pH3 with citric acid after, handled 3 hours at 100 ℃, modulate the acid degradation thing of this fucoidan.With this water degradation product with CellulofineGCL-300 or Cellulofine GCL-25 gel filtration, the classification molecular weight, be classified as molecular weight and surpass 25000 (A components), 25000～above 10000 (B components), 10000～above 5000 (C components), 5000～above 2000 (D components), 2000～above 500 (E components), (F component) below 500.Further these components and acid degradation thing are carried out lyophilization after the desalination respectively, each classification thing of preparation acid degradation thing and acid degradation thing.Reference example 9

Commercially available salt Tibetan Nemacystus decipiens (Sur.) Kuck is belonged to 5kg mix, cut off with shears with 20 liters of ethanol.Use filter paper filtering after placing for 1 evening, be suspended in 12.5 premium on currency, handled 2 hours at 95 ℃ with residue obtained.With treatment fluid with filter paper filtering after, add 2.5% the cetylpyridinium chloride solution 2600ml contain 350mM sodium chloride, placed 3 days.Discarded supernatant will precipitate the part centrifugalize, discarded supernatant.After in the gained precipitation, adding the sodium chloride of 2.5 liters of 350mM, use the homogenizer homogenize, centrifugalize.Repeat this washing operation 3 times.After in the gained precipitation, adding the 400mM sodium chloride of 400ml, use the homogenizer homogenize, add ethanol and make it to reach 80%, stir and use filter paper filtering after 30 minutes.In residue obtained, add saturated sodium-chloride 80% alcoholic solution of 500ml, afterwards, use the homogenizer homogenize, add the saturated sodium-chloride alcoholic solution and make it to reach 1 liter, stir and use filter paper filtering after 30 minutes.This washing operation (is generally 5 times) till the absorbance of 260nm reaches 0 up to filtrate repeatedly.After in the residue obtained sodium chloride solution that is dissolved in 1.5 liters of 2M, insoluble matter is removed in centrifugalize, makes it flow out the 100mlDEAE Cellulofine A-800 post of the sodium chloride equilibrating of using 2M in advance.After effusive component is concentrated into 2 liters by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000, solution is replaced into 2mM sodium chloride by ultrafiltration.This solution centrifugal is separated,, obtain the fucoidan that the 22.9g Nemacystus decipiens (Sur.) Kuck belongs to origin the lyophilization of gained supernatant.Reference example 10

(1) exsiccant Eucheuma gelatinosum 50g is cut off with shears, it is suspended in 80% ethanol of 500ml the back stirred 3 hours, use filter paper filtering at 25 ℃.Be suspended in 1 liter of 30mM sodium phosphate buffer (PH6.5) that contains 100mM sodium chloride residue obtained, 95 ℃ handle 2 hours after, with the stainless steel strainer filtering of aperture 106 μ m.In gained filtrate, add above-mentioned phosphate buffer, reach 3 liters, add the 5g active carbon, 25 ℃ stir a night after, centrifugalize.The gained supernatant carries out the solution exchange by ultrafilter after being concentrated into 200ml by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000, is exchanged for the 10mM sodium chloride solution.After removing insoluble matter in the solution by centrifugalize, lyophilization obtains the sulfated polysaccharides component 2.3g of Eucheuma gelatinosum origin.

(2) use the method for putting down in writing among the reference example 10-(1), Congjiang hedge belongs to 50g and prepares the sulfated polysaccharides 4.4g that the river hedge belongs to origin.In addition, similarly prepare the sulfated polysaccharides 1.0g of ペ テ ロ Network ラ デ ィ ァ origin from ペ テ ロ Network ラ デ ィ ァ キ ャ ピ ラ セ ラ.

(3)-1. stirred 3 hours at 25 ℃ after commercially available dry Macrocystis powder 1kg being suspended in 10 liter of 80% ethanol, use filter paper filtering.Be suspended in 20 liters of 30mM sodium phosphate buffers (PH6.5) that contain 100mM sodium chloride residue obtained, 95 ℃ handle 2 hours after, with the stainless steel strainer filtering of aperture 106 μ m.In gained filtrate, add the 100g active carbon, 2.4 liters of ethanol, the alginate lyase K of 6000U, 25 ℃ stir 22 hours after, centrifugalize.The gained supernatant is removed insoluble matter by centrifugalize after being concentrated into 1.2 liters by the ultrafilter that is equipped with the hollow fibre of getting rid of molecular weight 100,000, places 24 hours at 5 ℃.Remove the precipitation of generation by centrifugalize, the gained supernatant carries out the solution exchange with ultrafilter, is exchanged for the 100mM sodium chloride solution.After being cooled to this solution below 4 ℃, transfer to PH2.0 with hydrochloric acid, the precipitation of generation is removed in centrifugalize.With sodium hydroxide with the PH of gained supernatant modulation 8.0, be concentrated into 2 liters after, be replaced into the sodium chloride solution of 20mM with ultrafilter.After removing insoluble matter in this solution by centrifugalize, lyophilization obtains the dry thing 41g of the sulfated polysaccharides component of Macrocystis origin.

(3)-2. above-mentioned lyophilization thing 6g is dissolved among 20mM imidazoles-hydrochloride buffer (PH6) 600ml that contains 100mM sodium chloride, it is added on 5 liters of DEAE-Cellulofine A-800 posts using the same buffer equilibrating in advance, after cleaning with 10 liters of same buffer, carry out gradient elution with 100～1600mM sodium chloride solution.The liquid measure of using in the eluting is 13 liters, collects with every bottle of 500ml.In the elution fraction, to dialyse with the 500ml pure water with near the sodium chloride elution fraction 250mM, 530mM and the 700mM respectively, lyophilization with lyophilization product difference called after DEAE fraction 33, DEAE fraction 37, DEAE fraction 40, obtains 57mg, 24mg and 62mg respectively.Reference example 11

Stichopus japonicus (マ Na マ コ) 5kg is disintegrated, remove internal organs, collect body wall.Add 500ml acetone with respect to body wall weight in wet base 200g, handle after-filtration with homogenizer, with residue with washing with acetone till not having coloring material on it.With this residue suction dried, obtain the dry thing of 140g.What add 0.4M in this drying thing is 2.8 liters of saline,, filters after 1 hour 100 ℃ of processing, and residue is fully clean with the saline solution of 0.4M, obtains 3.7 liters of extracts.In this extract, add pyrisept till not generating precipitation, filter and collect the precipitation that generates.Recentrifuge separates after this precipitation being suspended in the saline solution of 0.4M, adds 1 liter of 4M saline solution in the gained precipitation, after handling with homogenizer, adds 4 liters of ethanol in the time of stirring, stirs after 1 hour, filters, and obtains precipitating.For this precipitation, repeating so-called is 0 up to supernatant at the absorbance of 260nm with its step that is suspended in after-filtration in 80% ethanol.Gained precipitation is suspended in 2 liters of 2M saline solutions, removes insoluble matter by centrifugalize, supernatant is by being equipped with the ultrafiltration apparatus ultrafiltration of the film of getting rid of molecular weight 30,000, and fully after the desalination, lyophilization obtains the fucoidan 3.7g of Stichopus japonicus origin.Reference example 12

After being suspended in 500mg agar powder (nakelaitesque Co., Ltd. system) in the 100ml distilled water, heating makes the agar dissolving.Afterwards, be cooled to 45 ℃, 45 ℃ of insulations.

In this agar solution, add X50 β beta-Agarase buffer (FMC society system: be attached in the β beta-Agarase) 2ml, 1U/ μ l β beta-Agarase (FMC society system) 100 μ l.This solution after 24 hours, is added the ethanol of 2.5 times of amounts 45 ℃ of insulations, and precipitation is reclaimed in cooling back centrifugalize.Should precipitate drying, be dissolved in the distilled water of 20ml.With this lysate lyophilization, prepare pulverous agaropectin component.Reference example 13

(1) the dry thalline 10g of Spirullina (Spirulina platensis) is suspended in the 100ml chloroform, filters, reclaim the operation of indissolvable component and carry out repeatedly 5 times.Afterwards, be suspended in the 100ml ethanol, filter, the operation of reclaiming indissolvable component repeats 3 times.From the indissolvable component that this operation obtains, remove ethanol fully, it is suspended in the distilled water of 100ml.With this suspension 60 ℃ the insulation 1 hour after, centrifugalize obtains supernatant.This supernatant is further filtered, in filtrate, add the ethanol of 2.5 times of amounts, after-20 ℃ of coolings, obtain precipitation in the low-temperature centrifugation separation.In distilled water, lyophilization prepares the component of the sulfur-bearing acidify polysaccharide of pulverous Spirullina origin with this resolution of precipitate.

(2) (sell the merchant: (strain) Spirullina institute) 20g puts into homogenizer (Japanese smart machine society system), adds the acetone of 400ml, 8000rpm homogenize 10 minutes with dry Spirullina powder.With the homogenate filter paper filtering, obtain residue.With the residue washing with acetone, repeat this operation 3 times with the mode identical, obtain the residue that acetone is cleaned with aforementioned operation.The residue mode same with washing with acetone that acetone is cleaned, with 90% washing with alcohol 4 times, 80% washing with alcohol 4 times obtains the clean residue of ethanol.

In the residue that ethanol is cleaned, add 600ml and contain 100mM sodium chloride and 10% alcoholic acid 30mM phosphate buffer (pH7.0), stirring at room 18 hours.This mixture 10000rpm centrifugalize 40 minutes, is obtained supernatant.With the insoluble matter filter paper filtering of sneaking in the supernatant, obtain thick extract (filtrate).After the thick extract of gained is concentrated into 300ml by the ultrafiltration apparatus that is equipped with the hollow fibre of getting rid of molecular weight 10,000, adds 2 liters and carry out ultrafiltration when containing 10% alcoholic acid 100mM sodium chloride.Afterwards, carry out solvent exchange, obtain 240ml Spirullina high molecular component with the 10mM imidazoles-hydrochloride buffer (pH7.0) that contains 10% ethanol and 50mM sodium chloride.

The Spirullina high molecular component is added to the DEAE-Cellulofine A-800 post of 10mM imidazoles-hydrochloride buffer (pH7.0) equilibrating that is contained 10% ethanol and 50mM sodium chloride (on the φ 3 * 14.2cm), after with same buffer 360ml pillar being cleaned, carry out gradient elution to the sodium chloride of 2M (200ml) with 0.05M (200ml).Eluent is with every bottle of 10ml fractional distillation.In the component that eluting goes out, respectively with fraction No.14～30 called after Spirullina sulfated polysaccharides component-I (SSP-I), fraction No.69～77 called after Spirullina sulfated polysaccharides component-II (SSP-II), fraction No.78～83 called after Spirullina sulfated polysaccharides component-III (SSP-III), fraction No.84～99 called after Spirullina sulfated polysaccharides component-IV (SSP-IV).SSP-I, SSP-II, SSP-III and SSP-IV are fully dialysed with distilled water respectively, and lyophilization obtains 200mg, 260mg, 100mg and 60mg respectively.

(3) the dry thalline 10g with Chlorella (Chlorella vulgaris) is suspended in the 100ml chloroform, and the filtered and recycled indissolvable component repeats this operation 3 times.Afterwards, it is suspended in the 100ml ethanol, filters, reclaim indissolvable component, repeat this operation 3 times.From the indissolvable component that this operation obtains, remove ethanol fully, it is suspended in the 100ml distilled water.This suspension, is filtered after 1 hour 60 ℃ of insulations.In filtrate, add the ethanol of 2.5 times of amounts, after-20 ℃ of coolings, separate, obtain precipitation at low-temperature centrifugation.In distilled water, lyophilization prepares the component that contains sulfated polysaccharides of pulverous Chlorella origin with this resolution of precipitate.

(4) (sell the merchant: (strain) Chlorella Center) 20g puts into homogenizer (Japanese smart machine society system), adds the acetone of 400ml, 8000rpm homogenize 10 minutes with dried pellet Trentepohlia powder.With the homogenate filter paper filtering, obtain residue.With the residue washing with acetone, repeat this operation 3 times with the mode identical, obtain the residue that acetone is cleaned with aforementioned operation.The residue mode same with washing with acetone that acetone is cleaned, with 90% washing with alcohol 4 times, 80% washing with alcohol 4 times obtains the clean residue of ethanol.

In the residue that ethanol is cleaned, add 600ml and contain 100mM sodium chloride and 10% alcoholic acid 30mM phosphate buffer (pH7.0), stirring at room 18 hours.This mixture 10000rpm centrifugalize 40 minutes, is obtained supernatant.With the insoluble matter filter paper filtering of sneaking in the supernatant, obtain thick extract (filtrate).After the thick extract of gained is concentrated into 310ml by the ultrafiltration apparatus that is equipped with the hollow fibre of getting rid of molecular weight 10,000, adds 3 liters and carry out ultrafiltration when containing 10% alcoholic acid 100mM sodium chloride.Afterwards, carry out solvent exchange, obtain 203ml Chlorella high molecular component with the 10mM imidazoles-hydrochloride buffer (pH7.0) that contains 10% ethanol and 50mM sodium chloride.

The Chlorella high molecular component is added to the DEAE-Cellulofine A-800 post of 10mM imidazoles-hydrochloride buffer (pH7.0) equilibrating that is contained 10% ethanol and 50mM sodium chloride (on the φ 3 * 14.2cm), after with same buffer 297ml pillar being cleaned, carry out gradient elution to the sodium chloride of 2M (200ml) with 0.05M (200ml).Eluent is with every bottle of 10ml fractional distillation.In the component that eluting goes out, respectively with fraction No.63～68 called after Chlorella sulfated polysaccharides component-I (CPS-I), fraction No.69～75 called after Chlorella sulfated polysaccharides component-II (CPS-II).CPS-I and CPS-II are fully dialysed with distilled water respectively, and lyophilization obtains 140mg and 200mg respectively.

(5) (Altemisia princeps pampan: this Chinese prescription of slope hall system) the Radix Artemisia ordosicae powder 10g after the pulverizing is suspended in the 100ml chloroform, and the filtered and recycled indissolvable component repeats this operation 3 times with commercially available Radix Artemisia ordosicae.Afterwards, it is suspended in the 100ml ethanol, filters, reclaim indissolvable component, repeat this operation 5 times.From the indissolvable component that this operation obtains, remove ethanol fully, it is suspended in the 100ml distilled water.This suspension, is filtered after 1 hour 60 ℃ of insulations.In filtrate, add the ethanol of 2.5 times of amounts, after-20 ℃ of coolings, separate, obtain precipitation and Radix Artemisia ordosicae supernatant component at low-temperature centrifugation.In distilled water, lyophilization prepares the component that contains sulfated polysaccharides of pulverous Radix Artemisia ordosicae origin with this resolution of precipitate.

(6) (sell the merchant: this Chinese prescription of slope hall) 50g puts into homogenizer (Japanese smart machine society system), adds the acetone of 500ml, 8000rpm homogenize 10 minutes with dry Folium Artemisiae Argyi.With the homogenate filter paper filtering, obtain residue.Repeat aforesaid operations 2 times, gained 100g Folium Artemisiae Argyi residue is put into homogenizer, add the acetone of 500ml, 8000rpm homogenize 10 minutes.With the homogenate filter paper filtering, obtain residue.Repeat this operation 4 times, obtain the residue that acetone is cleaned.The residue mode same with washing with acetone that acetone is cleaned, with 90% washing with alcohol 4 times, 80% washing with alcohol 4 times obtains the clean residue of ethanol.

In the residue that ethanol is cleaned, add 5 liters and contain 100mM sodium chloride and 10% alcoholic acid 30mM phosphate buffer (pH8.0), stirring at room 19 hours.With this mixture filter paper filtering, obtain thick extract (filtrate).After the thick extract of gained is concentrated into 2 liters by the ultrafiltration apparatus that is equipped with the hollow fibre of getting rid of molecular weight 10,000, adds 10 liters and carry out ultrafiltration when containing 10% alcoholic acid 100mM sodium chloride.Afterwards, be concentrated into 500ml, carry out solvent exchange with the 10mM imidazoles-hydrochloride buffer (pH7.0) that contains 10% ethanol and 50mM sodium chloride, this liquid is moved in the beaker, add the 1g active carbon, in stirring at room after 40 minutes, 10000rpm centrifugalize 40 minutes.With the active carbon filter paper filtering of sneaking in the supernatant, remove.Like this, obtain Folium Artemisiae Argyi high molecular component 560ml.

The Folium Artemisiae Argyi high molecular component is added to the DEAE-Cellulofine A-800 post of 10mM imidazoles-hydrochloride buffer (pH7.0) equilibrating that is contained 10% ethanol and 50mM sodium chloride (on the φ 3.5 * 31cm), after with same buffer 940ml pillar being cleaned, carry out gradient elution to the sodium chloride of 2M (600ml) with 0.05M (600ml).Eluent is with every bottle of 10ml fractional distillation.In the component that eluting goes out, respectively with fraction No.180～202 called after Folium Artemisiae Argyi acidic polysaccharose components (YAP), fraction No.203～270 called after Folium Artemisiae Argyi sulfated polysaccharides components (YSP).YAP is fully dialysed with distilled water, and lyophilization obtains 250mg.

For with the further classification of Folium Artemisiae Argyi sulfated polysaccharides component, Folium Artemisiae Argyi sulfated polysaccharides component is dialysed with 3 liters of 10mM imidazoles-hydrochloride buffers (pH7.0) that contain 10% ethanol and 100mM sodium chloride.The sulfated polysaccharides component (327ml) of dialysis is added to DEAE-Cellulofine A-800 post with same buffer equilibrating (on the φ 3 * 14.2cm).After pillar cleaned with the buffer of 273ml, carry out gradient elution to the sodium chloride of 2M (200ml) with 0.1M (200ml).Eluent is with every bottle of 5ml fractional distillation.In the component that eluting goes out, respectively with fraction No.140～154 called after Folium Artemisiae Argyi sulfated polysaccharides component-I (YSP-I), fraction No.155～200 called after Folium Artemisiae Argyi sulfated polysaccharides component-II (YSP-II).YSP-I and YSP-II are fully dialysed with distilled water, and lyophilization obtains 20mg and 130mg respectively.

In YSP-II (119.4mg), add 10% ethanol 59.7ml and contain the 10mM imidazoles-hydrochloride buffer (pH7.0) of 0.2M sodium chloride, at one night of stirring at room, dissolving.YSP-II is added to DEAE-Cellulofine A-800 post with same buffer equilibrating (on the φ 2.5 * 10.2cm).After pillar cleaned with the buffer of 200ml, carry out gradient elution to the sodium chloride of 1M (100ml) with 0.2M (100ml).Eluent is with every bottle of 5ml fractional distillation.In the component that eluting goes out, respectively with fraction No.54～70 called after Folium Artemisiae Argyi sulfated polysaccharides component-II-2 (YSP-II-2), fraction No.71～90 called after Folium Artemisiae Argyi sulfated polysaccharides component-II-3 (YSP-II-3), fraction No.91～120 called after Folium Artemisiae Argyi sulfated polysaccharides component-II-4 (YSP-II-4).YSP-II-2, YSP-II-3 and YSP-II-4 are fully dialysed with distilled water respectively, and lyophilization obtains 39.5mg, 61mg and 57.3mg respectively.

(7) commercially available edible Fructus Momordicae charantiae is pulverized the ground product lyophilization that obtains with blender, obtain the dry thing of Fructus Momordicae charantiae.Dry thing 10g is suspended in the 100ml chloroform with Fructus Momordicae charantiae, and the filtered and recycled indissolvable component repeats this operation 5 times.Afterwards, it is suspended in the 100ml ethanol, filters, reclaim indissolvable component, repeat this operation 3 times.From the indissolvable component that this operation obtains, remove ethanol fully, it is suspended in the 100ml distilled water.This suspension, is filtered after 1 hour 60 ℃ of insulations.In filtrate, add the ethanol of 2.5 times of amounts, after-20 ℃ of coolings, separate, obtain precipitation at low-temperature centrifugation.In distilled water, lyophilization prepares pulverous component that contains sulfated polysaccharides with this resolution of precipitate.

(8) the mesophyll part of recovery transparence from 5 pieces of commercially available Folium Aloes, lyophilization.0.481g is suspended in the 100ml distilled water with this Aloe mesophyll lyophilization thing.This suspension, is filtered after 1 hour 60 ℃ of insulations.In filtrate, add the ethanol of 2.5 times of amounts, after-20 ℃ of coolings, separate, obtain precipitation at low-temperature centrifugation.In distilled water, lyophilization prepares the component that contains sulfated polysaccharides of pulverous Aloe mesophyll origin with this resolution of precipitate.

On the other hand, after will reclaiming the residual green liquid surface portion of transparent mesophyll part and pulverize with said method, lyophilization.3.43g is suspended in the 100ml chloroform with this lyophilization thing, and the filtered and recycled indissolvable component repeats this operation 3 times.Afterwards, it is suspended in the 100ml ethanol, filters, reclaim indissolvable component, repeat this operation 3 times.From the indissolvable component that this operation obtains, remove ethanol fully, it is suspended in the 100ml distilled water.This suspension, is filtered after 1 hour 60 ℃ of insulations.In filtrate, add the ethanol of 2.5 times of amounts, after-20 ℃ of coolings, separate, obtain precipitation at low-temperature centrifugation.In distilled water, lyophilization prepares the component that contains sulfated polysaccharides of pulverous Folium Aloe surface origin with this resolution of precipitate.Reference example 14

(1) D-(+)-glucose 200mg (1.1mmol) is dissolved among the pyridine 10ml, adds pyridine sulfur trioxide complex (PyrSO in room temperature ₃: Tokyo changes into) behind the 1.05g (6.6mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.With the reactant liquor dilute with water, with saturated baryta water PH is transferred near the neutrality, then drying under reduced pressure.In the gained concentrate, add entry, drying under reduced pressure once more once more.Repeat once this operation again.Add low amounts of water in the gained concentrate, the centrifugal barium sulfate precipitate of removing is added to cation exchange column [Amberlite IRA-120 (Na with the gained supernatant ⁺) (Organo)] on, its result, will be from post effusive component concentrating under reduced pressure, obtain sulphation D-(+)-glucose sodium salt 700mg.

(2) D-(+)-galactose 240mg (1.3mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃1.05g (6.6mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation D-(+)-galactose sodium salt 406mg.

(3) D-(+)-mannose 200mg (1.3mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃1.05g (6.6mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation D-(+)-mannose sodium salt 700mg.

(4) maltose 205mg (0.57mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃Behind the 816mg (5.2mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation maltose sodium salt 520mg.

(5) maltotriose 200mg (0.4mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃Behind the 700mg (4.4mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation maltotriose sodium salt 420mg.

(6) trehalose 250mg (0.73mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃1.1g (7mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation trehalose sodium salt 750mg.

(7) lactose 222mg (0.62mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃Behind the 785mg (4.9mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation lactose sodium salt 406mg.

(8) sucrose 220mg (0.62mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃Behind the 785mg (4.9mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain Sodium sucrose sulfate salt 481mg.

(9) lactulose 370mg (1.08mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃1.38g (8.8mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation lactulose sodium salt 406mg.

(10) 6-(.alpha.-D-galactosido)-D-glucose. 379mg (0.9mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃1.43g (9.0mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation 6-(.alpha.-D-galactosido)-D-glucose. sodium salt 950mg.

(11) D-(+)-xylose 150mg (1.0mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃Behind the 770mg (4.8mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation D-(+)-xylose sodium salt 350mg.

(12) 2-deoxyglucose 200mg (1.2mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃Behind the 920mg (5.8mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation 2-deoxyglucose sodium salt 500mg.

(13) D-sorbitol 150mg (0.83mmol) is dissolved among the pyridine 10ml, adds PyrSO in room temperature ₃Behind the 955mg (6mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation D-sorbitol sodium salt 570mg.

(14) cellobiose 147mg (0.43mmol) is dissolved among the dimethyl sulfoxide 5ml, adds PyrSO in room temperature ₃Behind the 657mg (4.13mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation cellobiose sodium salt 230mg.

(15) dextrinose 62mg (0.18mmol) is dissolved among the pyridine 5ml, adds PyrSO in room temperature ₃Behind the 275mg (1.73mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation dextrinose sodium salt 162mg.

(16) turanose 293mg (0.86mmol) is dissolved among the pyridine 5ml, adds PyrSO in room temperature ₃Behind the 1310mg (8.22mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation turanose sodium salt 835mg.

(17) パ ラ チ ノ one ス 315mg (0.875mmol) is dissolved among the pyridine 5ml, adds PyrSO in room temperature ₃1.34g (8.4mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation パ ラ チ ノ one ス sodium salt 845mg.

(18) α-D-talose 56mg (0.31mmol) is dissolved among the pyridine 5ml, adds PyrSO in room temperature ₃Behind the 300mg (1.9mmol),, stirred 1 hour at 60 ℃ at stirring at room number minute.Below, carry out same operation according to reference example 14-(1), obtain sulphation D-talose sodium salt 150mg.

(19) full acetylated alpha-cyclodextrin 7g is handled with acetic anhydride and vitriolic mixed liquor (49: 1), obtain full acetylated MALTOHAXAOASE, it is deacetylated by Feldalat NM (NaOMe) in methanol, obtain MALTOHAXAOASE 1.5g.MALTOHAXAOASE 79mg (0.83mmol), piperidines sulphuric acid 1.33g are dissolved among dimethyl sulfoxide (DMSO) 5ml, stirred 2 hours at 80 ℃.After the reactant liquor cooling, with the dialyzer dialysis of getting rid of molecular weight 1,000 2 days.The gained dialyzed solution is added to cation exchange column [Amberlite IRA-120 (Na ⁺) (Organo)] on, its result, will be from post effusive component concentrating under reduced pressure, obtain Maltohexaose sulfate sodium salt 167mg.

(20) full acetylated beta-schardinger dextrin-2.2g is handled with acetic anhydride and vitriolic mixed liquor (49: 1), obtain full acetylated Fructus Hordei Germinatus seven sugar, it is deacetylated by Feldalat NM (NaOMe) in methanol, obtain Fructus Hordei Germinatus seven sugared 0.5g.Fructus Hordei Germinatus seven sugared 20mg (0.83mmol), pyridine sulphuric acid 325mg are dissolved among the DMSO 5ml, stirred 2 hours at 80 ℃.Afterwards, carry out same operation, obtain sulphation Fructus Hordei Germinatus seven sugared sodium salt 45.6mg according to reference example 14-(19).

(21) with full acetylated MALTOHAXAOASE in dichloromethane, in the presence of Tritox, potassium carbonate, stir, obtain the ィ ミ デ one ト body of acetylation MALTOHAXAOASE.The ィ ミ デ one ト body of acetylation MALTOHAXAOASE and dodecanol in dichloromethane, are reacted as catalyst with trifluoromethanesulfonic acid trimethyl silyl ester, with the deacetylated dodecyl MALTOHAXAOASE that obtains of gained reactant.Dodecyl MALTOHAXAOASE 370mg (0.32mmol) is dissolved among the DMSO10ml, after 2 hours, carries out same operation according to reference example 14-(19) below, obtain sulphation dodecyl MALTOHAXAOASE sodium salt 700mg 80 ℃ of stirrings.

(22) starch 276mg is dissolved among the DMSO 10ml, adds PyrSO in room temperature ₃2.76g after, stirred 2 hours at 80 ℃.With after the reactant liquor cooling, add acetone, the indissolvable component of generation with methanol wash for several times after, dilute with water is added to cation exchange column [Amberlite IRA-120 (Na ⁺) (Organo)] on.Its result, will be from post effusive component concentrating under reduced pressure, obtain sulphation starch sodium salt 350mg.

(23) カ one De ラ Application 111mg is dissolved among the DMSO 5ml, adds PyrSO in room temperature ₃1.11g after, stirred 2 hours at 80 ℃.With after the reactant liquor cooling, add acetone, the indissolvable component dilute with water of generation, be neutralized near the PH neutrality with saturated aqueous sodium carbonate after, with the dialyzer dialysis of getting rid of molecular weight 1,000 1 day.The gained dialyzed solution is added to cation exchange column [Amberlite IRA-120 (Na ⁺) (Organo)] and go up after, obtain sulphation カ one De ラ Application sodium salt 180mg by drying under reduced pressure.

(24) pectin 267mg is dissolved among the DMSO 5ml, adds PyrSO in room temperature ₃2.67g after, stirred 2 hours at 80 ℃.After the reactant liquor cooling, carry out same operation according to reference example 14-(23), obtain sulphation pectin sodium salt 384mg.

Embodiment 1

(1) (CCL171: big Japanese pharmacy society system, code.02-021) 500 μ l are added in the 48 porocyte culture plates, so that concentration is 1 * 10 to contain the MRC-5 cell that suspends in the DME culture medium of 10% hyclone ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, culture medium is exchanged for the DME culture medium that contains 1% hyclone.Afterwards, interpolation is as the fucoidan of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of record among the reference example 1-(1) of sample, make its ultimate density reach 1,10,100 μ g/ml respectively, after cultivating 24 hours again, reclaim culture medium, use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit (Funakoshi society system, Code.RS-0641-00), measure the HGF amount in the culture medium.

In contrast, the distilled water of use and sample equivalent.The HGF amount 7.2ng/ml of contrast should be worth as 100%, and it is as shown in table 1 that each sample adds the HGF generation of distinguishing.In addition, this experiment is all carried out 2 times, gets its meansigma methods.

Table 1

The fucoidan (μ g/ml) of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin	HGF generation (%)
		????????????0 ????????????1 ????????????10 ????????????100	????100 ????214 ????339 ????339

Add the fucoidan group of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin and compare with the matched group that adds distilled water, the generation of HGF obviously increases.In addition, compare with interpolation heparin or low molecular weight heparin, because the generation of HGF significantly increases, therefore, the fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin with up to the present be identified the low molecular weight heparin of inducing the heparin that produces HGF or mean molecule quantity about 5000 and compare and have higher promotion HGF and produce active.

(2) with the same condition of embodiment 1-(1) under, measure component I, component I I, component III respectively with the method preparation of reference example 1-(2) record, with the 7-12SFd-F of the method preparation of reference example 2 record, with the 6-2S of the method preparation of reference example 4, prepare the fucoidan of Thallus Laminariae origin and the HGF generation inducing action of the fucoidan of the Fucus Vesiculosus origin that the method put down in writing with reference example 7 prepares with the method for reference example 6 records.Its result is shown in table 2～4.

Table 2

Sample concentration HGF generation

(μg/ml)??????????(％)

Component I 1 167

100??????????????234

Component I I 1 208

100??????????????359

Component III 1 146

100 291 (the HGF generation of contrast is 8.3ng/ml).

Table 3

Sample concentration HGF generation

(μ g/ml) be the fucoidan 1 148 of Fucus Vesiculosus origin (%)

10?????????246

The fucoidan 1 179 of 100 335 Thallus Laminariae origin

10?????????250

100????????291

7-12SFd-F????????????????1??????????149

10?????????276

100 339 (the HGF generation of contrast is 8.6ng/ml).

Table 4

Sample concentration HGF generation

(μg/ml)????(％)

6-2S???????????????????10????????112

100???????246

(the HGF generation of contrast is 9.9ng/ml).

Can confirm ガ go メ Thallus Laminariae (Thallus Eckloniae) origin fucoidan fraction promptly, the 7-12SFd-F of the fucoidan of U-fucoidan, F-fucoidan, Fucus Vesiculosus origin, the fucoidan of Thallus Laminariae origin, F-fucoidan origin, the 6-2S of U-fucoidan origin have strong HGF respectively and produce inducing action.In addition, can confirm that the acid degradation thing of fucoidan, reference example 8 records of Ascophyllum nodosum origin of fucoidan, reference example 7 records of fucoidan, the Macrocystis origin of the Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record and component A～F also have strong HGF respectively and produce inducing action.

(3)-1. 2% solution of the fucoidan of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin that will prepare with the method for reference example 1-(1) record transfers to PH3 with citric acid or sulphuric acid, respectively at 100 ℃ of heating their hydrolyzed solution of preparation after 30 minutes, after 1 hour, after 2 hours, after 4 hours, use and measure its HGF with the same condition of embodiment 1-(1) and produce inducing action.In addition, sample uses 10 times of diluents of acid degradation liquid.

Table 5

Sample	Heat time heating time	HGF generation (%)
			Degradation solution not		????448
The sulphuric acid degradation solution	30 minutes 1 hour 2 hours 4 hours	????364 ????385 ????368 ????345
			Degradation solution not		????480
The citric acid degradation solution	30 minutes 1 hour 2 hours 4 hours	????402 ????429 ????397 ????341

(the HGF generation of contrast is 8.6ng/ml).

(3)-2. heating 4 hour the handled thing of fucoidan in the presence of citric acid of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin that embodiment 1-(3)-is 1. prepared is by the gel filtration classification.

That is, will be with the pillar watering balanceization of 1.5 liters of fillings of TOYOPEARL HW40C, the fucoidan heat treated thing 10ml of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin is added on this post, divide the water eluting with flow velocity 1ml/.Initial 680ml former state flows away, and afterwards with a 14ml fractional distillation, obtains the gel filtration fraction of heat treated thing.

This fraction is analyzed with TLC (solvent: butyl acetate: acetic acid: water=3: 4: 3, detection agent sulphuric acid orcin), had more the drop sample, collect component 12～13,16～17,26～40 isogel filter component, lyophilization.The lyophilization thing of each component of gained is dissolved in water again, makes it to reach 100mg/ml, use and measure its HGF with the same condition of embodiment 1-(1) and produce inducing action.

Its result confirms that each fraction of component 12～13 and component 16～17 has HGF to produce inducing action.

The fraction of component 12～13 is carried out structure determine, its assay value is consistent with the international assay value that disclose chemical compound shown in the following formula of putting down in writing in No. 97/26896 (VIII), can confirm that the polymer of glucuronic acid and mannose has HGF generation induced activity.

(4) preparation commercially available dextran sulfate sodium solution (Sigma society system) is measured its HGF according to the method for embodiment 1-(1) and is produced inducing action.As shown in table 6, dextran sulfate sodium shows that HGF produces inducing action.

Table 6

Dextran sulfate sodium concentration HGF generation

(mean molecule quantity) (μ g/ml) (%)

10,000 1 588

10??????????655

100?????????787

8,000 1 395

10??????????573

100?????????695

5,000 1 398

10??????????421

100?????????565

(the HGF generation of contrast is 6.7ng/ml).

(5) prepare the solution (nacalaitesque society system) of commercially available λ-carrageenin, measure its HGF according to the method for embodiment 1-(1) and produce inducing action.As shown in table 7, λ-carrageenin shows that HGF produces inducing action.

Table 7

λ-carrageenin HGF generation

(μg/ml)??????????(％)

1??????????????152

10?????????????140

(the HGF generation of contrast is 13.4ng/ml).

(6)-1. prepare commercially available alginic acid solution (with the pure medicine of light society system: swelling), measure its HGF according to the method for embodiment 1-(1) and produce inducing action.As shown in table 8, alginic acid shows that HGF produces inducing action.

Table 8

Alginic acid HGF generation

(μg/ml)???????????(％)

1???????????????125

10??????????????154

100 327 (the HGF generation of contrast is 7.3ng/ml).

(6)-2. similarly, study the HGF generation induced activity of alginic acid (the pure medicine of swelling and light society system: sample 1.), alginic acid (the pure medicine of non-swelling and light society system: sample 2.), alginic acid (the pure medicine of 100～150cp and light society system: sample 3.), alginic acid (the pure medicine of 300～400cp and light society system: sample 4.), alginic acid (the pure medicine of 500～600cp and light society system: sample 5.) respectively.As shown in table 9,1.～5. sample all can induce the generation of HGF.From as can be known above, also have HGF as the alginic acid of acidic polysaccharose and produce induced activity.

Table 9

The generation of concentration HGF (%)

(μ g/ml) sample 1. sample 2. sample 3. sample 4. sample is 5.

10?????????154???????138???????115???????127???????104

100 327 158 184 152 187 (sample is 1., the HGF generation of matched group 2. is 7.3ng/ml, sample 3., 4., the HGF generation of matched group 5. is 6.7ng/ml).

(6)-3. similarly, the HGF of research pectic acid (nacalaitesque society system) produces induced activity.As shown in table 10, pectic acid is induced the generation of HGF.From as can be known above, also have HGF as the pectic acid of acidic polysaccharose and produce induced activity.

Table 10

Sample concentration HGF generation

(μ g/ml) be pectic acid 1 145 (%)

10?????????218

100 684 (the HGF generation of matched group is 9.9ng/ml).

(7) HGF of research salmon sperm dna (Nichiro society of Co., Ltd. system) produces induced activity.Adding DNA makes its ultimate density reach 1,10,100 μ g/ml.As shown in table 11, salmon sperm dna shows that HGF produces induced activity.

Table 11

The generation of sample concentration HGF

(μ g/ml) be salmon sperm dna 1 110 (%)

10?????????182

100 285 (the HGF generation of matched group is 9.9ng/ml).

(8) Nemacystus decipiens (Sur.) Kuck for preparing of preparation reference example 9,11 belongs to the fucoidan solution of origin, measures its HGF according to the method for embodiment 1-(1) and produces inducing action.As shown in table 12, each fucoidan shows that HGF produces inducing action.

Table 12 sample concentration HGF generation

(μ g/ml) be the fucoidan 1 282 of Stichopus japonicus origin (%)

10?????????356

100 495 Nemacystus decipiens (Sur.) Kuck belong to the fucoidan 1 218 of origin

10?????????279

100 307 (the HGF generation of matched group is 7.77ng/ml).

(9) sulfated polysaccharides (sample 1.) of the stone stilbene dish origin for preparing of preparation reference example 10, sulfated polysaccharides (sample 2.) that the river hedge belongs to origin, from the solution of the sulfated polysaccharides (sample 3.) of ペ テ ロ Network ラ デ ィ ァ origin, with the same quadrat method research HGF generation induced activity of embodiment 1-(1).Add sample make sample 1., ultimate density 3. is 1,10,100 μ g/ml, sample ultimate density 2. is 10,100 μ g/ml.As shown in table 13,1.～3. sample all can induce HGF to produce.

Table 13

The generation of sample concentration HGF

(μg/ml)??????????(％)

Sample 1. 1 100

10?????????????177

100????????????169

Sample 2. 10 117

100????????????198

Sample 3. 1 116

10?????????????207

100 228 (the HGF generation of matched group is 7.3ng/ml).

(10) according to the same quadrat method of embodiment 1-(1), the HGF of the fucoidan (sample 1.) of the Macrocystis origin that research reference example 10-(3) prepares, DEAE component 33 (sample 2.), DEAE component 37 (sample 3.), DEAE component 40 (sample 4.) produces induced activity.Adding each sample, to reach ultimate density be 1,10,100 μ g/ml.As shown in table 14,1.～4. sample all can induce the generation of HGF.

Table 14

The generation of concentration HGF (%) (μ g/ml) sample 1. sample 2. sample 3. sample is 4.

1????????138???????105???????235???????121

10???????167???????221???????253???????254

100 331 265 261 295 (the HGF generation of matched group is 11.5ng/ml).

(11) with the same condition of embodiment 1-(1) under, the generation induced activity of the HGF of the agaropectin of record, chondroitin sulfate B (biochemical industrial society system), chondroitin sulfate D (biochemical industrial society system) in the sulfated fucogalactan, reference example 12 of research reference example 3-(2) record.Adding each sample, to reach ultimate density be 1,10,100 μ g/ml.Shown in table 15～17, sulfated fucogalactan, agaropectin, chondroitin sulfate all can be induced the generation of HGF.

Table 15

The generation of sample concentration HGF

(μ g/ml) be sulfated fucogalactan 1 194 (%)

10??????????????355

100 429 (the HGF generation of matched group is 4.3ng/ml).

Table 16

The generation of sample concentration HGF

(μg/ml)???????????(％)

Agaropectin 1 117

10??????????????121

100 256 (the HGF generation of matched group is 6.7ng/ml).

Table 17

The generation of sample concentration HGF

(μg/ml)??????????(％)

Chondroitin sulfate B 1 117

10????????????121

100???????????256

Chondroitin sulfate D 10 119

100 114 (the HGF generation of matched group is 11.9ng/ml).

(12) according to the same quadrat method of embodiment 1-(1), sulfated polysaccharides, the sulfated polysaccharides of Chlorella origin, the sulfated polysaccharides of Radix Artemisia ordosicae origin, the Fructus Momordicae charantiae of the Spirullina origin that research reference example 13-(1), 13-(3), 13-(5), 13-(8) prepare respectively depends on the HGF of the sulfated polysaccharides of the sulfated polysaccharides of low sulphation polysaccharide, Aloe mesophyll origin and Folium Aloe surface thing origin to produce induced activity.Add the sulfated polysaccharides of Spirullina origin, the sulfated polysaccharides of Radix Artemisia ordosicae origin, make its ultimate density reach 1,10,100 μ g/ml.Add the sulfated polysaccharides of Chlorella origin, the sulfated polysaccharides of Fructus Momordicae charantiae origin, the sulfated polysaccharides of Aloe mesophyll origin, the sulfated polysaccharides of Folium Aloe surface thing origin, make its ultimate density reach 1,10,100,1000 μ g/ml.Shown in table 18～20, the generation that the sulfated polysaccharides of the sulfated polysaccharides of the sulfated polysaccharides of the sulfated polysaccharides of Spirullina origin, Chlorella origin, the sulfated polysaccharides of Radix Artemisia ordosicae origin, Fructus Momordicae charantiae origin, the sulfated polysaccharides of Aloe mesophyll origin and Folium Aloe surface thing origin all can be induced HGF.

Table 18

Sample	Concentration (μ g/ml)	The generation of HGF (%)
			The sulfated polysaccharides of Spirullina origin	????1 ????10 ????100	????149 ????293 ????398
The sulfated polysaccharides of Chlorella origin	????1 ????10 ????100 ????1000	????108 ????149 ????175 ????396

(the HGF generation of matched group is 7.9ng/ml)

Table 19

The generation of sample concentration HGF

(μ g/ml) be the sulfated polysaccharides 1 137 of Radix Artemisia ordosicae origin (%)

10????????????284

100 265 (the HGF generation of matched group is 12.7ng/ml).

Table 20

The generation of sample concentration HGF

(μ g/ml) be the sulfated polysaccharides 1 111 of Fructus Momordicae charantiae origin (%)

10?????????104

100????????133

The sulfated polysaccharides 1 111 of 1,000 190 Aloe mesophyll origin

10?????????125

100????????150

The sulphation 1 106 of 1,000 401 Folium Aloes surface thing origin

Polysaccharide 10 125

100????????120

1,000 328 (the HGF generation of matched group is 8.7ng/ml).

(13) according to the same quadrat method of embodiment 1-(1), the HGF of Spirullina fraction SSP-I (sample 1.), SSP-II (sample 2.), SSP-III (sample 3.) and SSP-IV (sample 4.) that research reference example 13-(2) prepares produces induced activity.Adding each sample makes ultimate density reach 1,10,100 μ g/ml.Shown in table 21,1.～4. sample all can induce the generation of HGF.

Table 21

The generation of concentration HGF (%) (μ g/ml) sample 1. sample 2. sample 3. sample is 4.

1?????????240???????165???????141???????218

10????????243???????152???????270???????282

100 302 212 280 351 (the HGF generation of matched group is 7.3ng/ml).

(14) according to the same quadrat method of embodiment 1-(1), the Chlorella extract CSP-I component (sample 1.) that research reference example 13-(4) prepares, the HGF of CSP-II (sample 2.) produce induced activity.Adding sample 1. makes ultimate density reach 10,100 μ g/ml.Adding sample 2. makes ultimate density reach 100 μ g/ml.Shown in table 22,1., 2. sample all can induce the generation of HGF.

Table 22

The generation of sample concentration HGF

(μg/ml)??????(％)

Sample 1. 10 111

100????????173

Sample is 100 175 (the HGF generation of matched group is 11.4ng/ml) 2..

(15) according to the same quadrat method of embodiment 1-(1), the HGF of Radix Artemisia ordosicae extract component YAP (sample 1.), the component YSP-I (sample 2.) that research reference example 13-(6) prepares, component YSP-II (sample 3.), component YSP-II-2 (sample 4.), component YSP-II-3 (sample 5.), component YSP-II-4 (sample 6.) produces induced activity.Adding each sample makes ultimate density reach 1,10,100 μ g/ml.Shown in table 23,24,1.～6. sample all can induce the generation of HGF.Can confirm that particularly component YSP-II (sample 3.), component YSP-II-3 (sample 4.), component YSP-II-4 (sample 5.) have strong HGF to produce induced activity.

Table 23

The generation of sample concentration HGF

(μg/ml)??????????(％)

Sample 1. 10 121

100????????????266

Sample 2. 1 104

10?????????????148

100????????????381

Sample 3. 1 328

10?????????????386

100 390 (the HGF generation of matched group is 11.5ng/ml).

Table 24

The generation of concentration HGF (%)

(μ g/ml) sample 4. sample 5. sample is 6.

1??????????152???????290???????226

10?????????462???????383???????322

100 475 321 314 (the HGF generation of matched group is 7.7ng/ml).

(16) according to the same quadrat method of embodiment 1-(1), study the sulphation maltose sodium salt that reference example 14 prepares, sulphation maltotriose sodium salt, sulphation lactose sodium salt, Sodium sucrose sulfate salt, sulphation trehalose sodium salt, the Glucose sulfate sodium salt, sulphation lactulose sodium salt, sulphation 6-(.alpha.-D-galactosido)-D-glucose. sodium salt, sulphation galactose sodium salt, sulphation mannose sodium salt, sulphation xylose sodium salt, sulphation 2-deoxyglucose sodium salt, the Sorbitol sulfate. sodium salt, sulphation cellobiose sodium salt, sulphation dextrinose sodium salt, sulfur sulphation turanose sodium salt, sulphation パ ラ チ ノ one ス sodium salt, sulphation talose sodium salt, the Maltohexaose sulfate sodium salt, sulphation Fructus Hordei Germinatus seven sugared sodium salts, sulphation dodecyl MALTOHAXAOASE sodium salt, sulphation starch sodium salt, sulphation カ one De ラ Application sodium salt, the HGF of sulphation pectin sodium salt produces induced activity.Adding each sample makes ultimate density reach 1,10,100 μ g/ml or 10,100,1000 μ g/ml or 100 μ g/ml respectively.In contrast, add the distilled water of equivalent.In addition, the HGF at concentration determination each the non-sulfuric acid sugar identical with each sulfated sugar produces induced activity.

Shown in table 25～34, the generation that sulfated oligosaccharide, sulphation monosaccharide can be induced HGF.In addition, do not induce the generation of HGF for each sugar of non-sulfuric acidization.

In addition, from the result of sulphation dodecyl MALTOHAXAOASE sodium salt as can be known, even sugar by lipid-modified, also keeps HGF to produce induced activity.

Table 25

The generation of sample concentration HGF

(μ g/ml) be sulphation maltose sodium salt 1 142 (%)

10?????????????273

100 439 sulphation maltotriose sodium salts 1 151

10?????????????286

100 387 (the HGF generation of matched group is 8.7ng/ml).

Table 26

The generation of sample concentration HGF

(μ g/ml) be sulphation lactose sodium salt 1 118 (%)

10????????????185

100 410 Sodium sucrose sulfate salt 1 173

10????????????355

100 501 Glucose sulfate sodium salts 1 178

10????????????377

100 864 (the HGF generation of matched group is 5.5ng/ml).

Table 27

The generation of sample concentration HGF

(μ g/ml) be sulphation trehalose sodium salt 1 277 (%)

10????????????447

100 421 (the HGF generation of matched group is 4.3ng/ml).

Table 28

The generation of sample concentration HGF

(μ g/ml) be sulphation galactose sodium salt 100 166 (the HGF generation of matched group is 12.7ng/ml) (%).

Table 29

The generation of sample concentration HGF

(μ g/ml) be sulphation mannose sodium salt 1 112 (%)

10??????????175

100 456 (the HGF generation of matched group is 6.2ng/ml).

Table 30

The generation of sample concentration HGF

(μ g/ml) be sulphation lactulose sodium salt 10 139 (%)

100?????????376

1,000 583 sulphation 6-(.alpha.-D-galactosido)-D-glucose. sodium salts 10 240

100?????????403

1,000 667 sulphation xylose sodium salts 10 110

100?????????112

1,000 284 sulphation 2-deoxyglucoses 10 127

Sodium salt 100 102

1,000 239 Sorbitol sulfate. sodium salts 10 112

100?????????203

1,000 335 (the HGF generation of matched group is 5.9ng/ml).

Table 31

The generation of sample concentration HGF

(μ g/ml) be sulphation cellobiose sodium salt 10 100 (%)

100???????????143

1,000 546 sulphation dextrinose sodium salts 10 134

100???????????301

1,000 477 sulphation turanose sodium salts 10 127

100???????????203

1,000 379 sulphation パ ラ チ ノ, one ス 10 132

Sodium salt 100 278

1,000 519 (the matched group HGF generation of sulphation cellobiose sodium salt is 11.1ng/ml, and the matched group HGF generation of sulphation dextrinose sodium salt is 11.3ng/ml, and the matched group HGF generation of sulphation turanose sodium salt is 8.6ng/ml.)

Table 32

The generation of sample concentration HGF

(μ g/ml) be sulphation talose sodium salt 10 112 (%)

100????????263

1,000 494 (the HGF generation of matched group is 9.5ng/ml).

Table 33

The generation of sample concentration HGF

(μ g/ml) be Maltohexaose sulfate sodium salt 10 407 (%)

100?????????571

1,000 761 sulphation Fructus Hordei Germinatus, seven sugared sodium salts 10 341

100?????????486

1,000 706 sulphation dodecyl Fructus Hordei Germinatus 10 289

Six sugared sodium salts 100 371

1,000 359 (the HGF generation of matched group is 8.15ng/ml).

The generation of table 34 sample concentration HGF

(μ g/ml) be sulphation starch sodium salt 10 781 (%)

100?????????864

1,000 804 sulphation カ, one De ラ Application sodium salt 10 359

100?????????503

1,000 617 sulphation pectin sodium salts 10 721

100?????????780

1,000 648 (the HGF generation of matched group is 8.15ng/ml).

Embodiment 2

(1), the inducing synergy of HGF of the fucoidan of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record and prostaglandin, IL-1 is studied according to the same quadrat method of embodiment 1-(1).

That is, add this fucoidan and PGE simultaneously ₁(with the pure medicine of light society system), IL-1 α (Genzyme society system), research HGF produces the synergy of induced activity.

Adding fucoidan makes its ultimate density reach 1,10,100 μ g/ml.Add PGE ₁Reach 0.1,1 μ M, IL-1 α reaches 1ng/ml.In contrast, add the distilled water of equivalent.

With add fucoidan sample or PGE respectively ₁, the generation during IL-1 α relatively, studied synergy.

Its result is shown in table 35,36.In table 35,36, the HGF generation that contrasts is decided to be 100%.Test is all carried out averaging for twice.Shown in table 35,36, can confirm by adding PGE simultaneously with fucoidan ₁Or IL-1 α, synergism has been induced in the generation of HGF.

The addition PGE of table 35 fucoidan ₁Addition (μ M)

(μg/ml)??????????????0???????0.1??????1

The generation of HGF (%)

0?????????????????100??????119??????157

1?????????????????195??????334??????415

10????????????????331??????517??????561

100???????????????382??????731??????682

Table 36 fucoidan addition IL-1 α addition (ng/ml)

(μg/ml)?????????????????0????????????????1

The generation of HGF (%)

0????????????????????100??????????????163

1????????????????????206??????????????421

10???????????????????350??????????????672

100 403 780 (the HGF generation of matched group is 9.1ng/ml).

(2), the inducing synergy of HGF of the 7-12SFd-F of reference example 2 and prostaglandin, IL-1 is studied according to the same quadrat method of embodiment 2-(1).That is, add 7-12SFd-F and PGE simultaneously ₁, IL-1 α, research HGF produces the synergy of induced activity.Adding 7-12SFd-F makes its ultimate density reach 1,10,100 μ g/ml.Further add PGE to the cell that has added 7-12SFd-F respectively ₁, IL-1 α.Add PGE ₁Reach 0.1,1 μ M, IL-1 α reaches 0.1,1ng/m1.As negative control, add the distilled water of equivalent.With add 7-12SFd-F or PGE respectively ₁, the generation during IL-1 α relatively, study its synergy.The generation of HGF is decided to be 100% with the negative control of only adding distilled water.Its result is shown in table 37/38.Test is all carried out averaging for three times.

Table 37

The addition PGE of 7-12SFd-F ₁Addition (μ M)

(μg/m1)????????????0????????0.1???????1

The generation of HGF (%)

0???????????????100???????123??????170

1???????????????120???????158??????216

10??????????????218???????309??????317

100 219 365 382 (the HGF generation of matched group is 5.1ng/m1).

Table 38

The addition IL-1 α addition (ng/m1) of 7-12SFd-F

(μg/m1)????????????0??????0.1??????1

The generation of HGF (%)

0??????????????100?????130?????151

1??????????????140?????168?????159

10?????????????191?????154?????208

100 203 255 222 (the HGF generation of matched group is 5.1ng/m1).

Embodiment 3

(1) the KG-1-C cell (sale of G1yoma:Human Science development financial group) that will be suspended in the DMEM culture medium that contains 10% hyclone adds 48 porocyte culture plates, and every hole 500 μ l reach 1 * 10 ⁵Cell/ml is at 37 ℃, 5%CO ₂After cultivating a night under existing, with the DMEM culture medium exchange that contains 1% hyclone.Afterwards, add sample, cultivated again 20 hours, reclaim culture medium, use the HGF ELISA test kit of embodiment 1 record, measure the amount of HGF in the culture medium.Tested sample ultimate density is respectively: the fucoidan that adds the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record reaches 1,10,100 μ g/ml, and heparin (with the pure medicine of light society system) reaches 1,10 μ g/ml.In addition, in contrast, add distilled water with sample equivalent.Test is all carried out 3 times, gets its meansigma methods.The result is shown in table 39.In table 39, the HGF generation of contrast is 100%.

All groups of cells of adding the fucoidan sample are significantly increased than the HGF generation of the matched group that adds distilled water.In addition, compare the also significantly increase of generation of HGF with the interpolation heparin.Hence one can see that, and this fucoidan has the heparin of HGF inducing action to show that higher HGF generation promotes active than up to the present confirming.

Table 39

The generation of sample solution concentration HGF

(μ g/ml) (%) ガ go メ Thallus Laminariae (Thallus Eckloniae) origin 1 194

Fucoidan 10 285

100?????????351

Calciparine/sodium salt 1 176

10 215 (the HGF generation of matched group is 4.4ng/ml).

(2) will be at the HL-60 cell of RPMI 1640 culture medium culturings that contain 10% hyclone (prebone marrow leukaemia cell: ATCC CCL-240) be suspended in the RPMI1640 culture medium that contains 1% hyclone and reach concentration 1 * 10 ⁵Cell/ml adds 48 porocyte culture plates with it, every hole 500 μ l.Afterwards, add the 12-O-myristoyl phorbol 13-acetate (TPA:Gibro BRL society system) of 10nM, and add tested sample simultaneously.Add the back and cultivate after 20 hours, reclaim culture medium, use HGF ELISA test kit, measure the amount of HGF in the culture medium.

Tested sample ultimate density is respectively: the fucoidan that adds the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record reaches 1,10,100 μ g/ml.Add heparin and reach 1,10 μ g/ml.In contrast, the distilled water of interpolation and sample equivalent.Test is all carried out 3 times, gets its meansigma methods.The result is shown in table 40.In table 40, the HGF generation of contrast is 100%.

All groups of cells of adding the fucoidan sample are significantly increased than the HGF generation of the matched group that adds distilled water.In addition, compare the also significantly increase of generation of HGF with the interpolation heparin.Hence one can see that, and this fucoidan has the heparin of HGF inducing action to show that higher HGF generation promotes active than up to the present confirming.

Table 40

The generation of sample solution concentration HGF

(μ g/ml) be the rock 1 191 of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin (%)

Algae is according to polysaccharide 10 342

100?????????490

Calciparine/sodium salt 1 140

10 189 (the HGF generation of matched group is 0.4ng/ml).

Embodiment 4

The MRC-5 Cell sap that is suspended in the DMEM culture medium that contains 10% hyclone is added 48 porocyte culture plates, and every hole 500 μ l reach 1 * 10 ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DMEM culture medium exchange that contains 1% hyclone.Afterwards, add sample, cultivated again 24 hours, reclaim culture medium, use HGF ELISA test kit, measure the amount of HGF in the culture medium.Further, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mM HEPES pH7.4,10mM EDTA, 0.1%Triton X100,1mM PMSF, 1 μ g/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.

Tested sample ultimate density is respectively: the fucoidan that adds the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record reaches 1,10,100 μ g/ml.In contrast, the distilled water of interpolation and sample equivalent.Test is all carried out 2 times, gets its meansigma methods.The result is shown in table 41.Shown in table 41, the HGF amount in the culture medium of this interpolation fucoidan interpolation group significantly increases in the mode that depends on fucoidan concentration than the amount in the matched group that adds distilled water.On the other hand, intracellular HGF amount reduces in the mode that exists with ... fucoidan concentration.Then, the inside and outside total HCF amount concentration of cell relies on ground increases.Hence one can see that, and this fucoidan shows the free motion active and promotion HGF from cell that promotes that HGF produces.

The rock algae HGF generation (ng/ hole) of table 41 ガ go メ Thallus Laminariae (Thallus Eckloniae) origin is according to total amount in the cell in polysaccharide (μ g/ml) culture medium

Contrast 4.2 5.7 9.8

1????????????????????9.3????????3.2??????12.5

10???????????????????15.9???????0.9??????16.8

100??????????????????16.9???????0.4??????17.3

Embodiment 5

(1) the MRC-5 Cell sap 500 μ l that will be suspended in the DME culture medium that contains 10% hyclone add 48 porocyte culture plates, reach 1 * 10 ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DME culture medium exchange that contains 1% hyclone.Afterwards, add sample, cultivate after 0,0.5,1,2,4,8,12,24 hour, reclaim culture medium, use HGF ELISA test kit, measure the amount of HGF in the culture medium.The fucoidan that adds the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record makes ultimate density reach 10 μ g/ml, in contrast, adds the distilled water with sample equivalent.The result is shown in table 42.Shown in table 42, fucoidan interpolation group significantly increases the generation of HGF in interdependent mode of time than matched group.Show that thus fucoidan has high HGF and produces the promotion activity, the generation of HGF increases along with the increase of time.

Table 42

Incubation time (hour)

0?????0.5???1?????2?????4?????8?????12????24

The generation of HGF (ng/ml) contrast 0.02 0.03 0.81 0.90 2.31 3.90 6.94 9.39 is added rock algae 0.19 1.90 5.75 6.87 9.04 15.9 20.1 31.3 according to polysaccharide

(2) (CCL171: big Japanese pharmacy society system, code.02-021) 500 μ l add 48 porocyte culture plates, reach 1 * 10 will to be suspended in the MRC-5 cell of the DME culture medium that contains 10% hyclone ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DMEM culture medium exchange that contains 1% hyclone.Afterwards, add sample, cultivate after 0,0.5,1,2,4,8,12,24,48,72 hour, reclaim culture medium, (Funakoshi society system code.RS-0641-00), is measured the amount of HGF in the culture medium to use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit.Further, after reclaiming culture medium, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mM HEPES pH7.4,10mM EDTA, 0.1%Triton X100,1mM PMSF, 1 μ/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.The fucoidan that adds the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record makes its ultimate density reach 10 μ g/ml.As negative control, add distilled water with sample equivalent.HGF concentration ratio negative control group in the culture medium of this fucoidan interpolation group significantly increases in interdependent mode of time.On the other hand, the intracellular HGF amount of this fucoidan interpolation group reduces by 4 hours from adding the back, but remains on certain low value thereafter.In the negative control group of adding distilled water, there is not such variation, show the tendency that increases usually.Show that thus effect and HGF that this fucoidan has free HGF from cell produce the promotion activity, the generation of HGF increases along with the increase of time.This result is shown in table 43～45.

The HGF amount over time in table 43 culture medium

0?????0.5????1?????2?????4?????8?????12????24????48????72

The generation of HGF (ng/ hole) contrast 0 0.11 0.19 0.29 1.07 1.56 2.34 3.46 7.45 10.5 is added rock algae 0.51 0.46 1.23 2.86 4.00 6.70 9.15 12.4 22.8 29.3 according to polysaccharide

The HGF amount over time in table 44 cell

Incubation time (hour)

0?????0.5???1?????2?????4?????8?????12????24????48????72

The generation of HGF (ng/ hole) contrast 0.51 0.52 0.66 0.56 0.71 0.63 0.86 0.73 1.02 1.20 is added rock algae 0.88 0.66 0.45 0.46 0.25 0.29 0.20 0.20 0.18 0.23 according to total HGF amount in polysaccharide table 45 culture medium and in the cell over time

Incubation time (hour)

0?????0.5???1?????2?????4?????8?????12????24????48????72

The generation of HGF (ng/ hole) contrast 0.51 0.63 0.85 0.84 1.78 2.19 3.19 4.19 8.47 11.6 is added rock algae 1.39 1.12 1.68 3.31 4.25 7.00 9.35 12.6 22.9 29.5 according to polysaccharide

(3) (CCL171: big Japanese pharmacy society system, code.02-021) 500 μ l add 48 porocyte culture plates, reach 1 * 10 will to be suspended in the MRC-5 cell of the DMEM culture medium that contains 10% hyclone ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DME culture medium exchange that contains 1% hyclone.Afterwards, add sample, cultivate after 0,0.5,1,2,4,8,12,24,48,72 hour, reclaim culture medium, (Funakoshi society system code.RS-0641-00), is measured the amount of HGF in the culture medium to use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit.Further, after reclaiming culture medium, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mM HEPES pH7.4,10mM EDTA, 0.1%Triton X100,1mM PMSF, 1 μ/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.Adding 7-12SFd-F makes its ultimate density reach 10 μ g/ml.As negative control, add distilled water with sample equivalent.HGF concentration ratio negative control group in the culture medium of this 7-12SFd-F interpolation group significantly increases in interdependent mode of time.On the other hand, the intracellular HGF amount of this 7-12SFd-F interpolation group reduces soon after the interpolation, but transfers increase again to afterwards.In the negative control group of adding distilled water, there is not such variation, show certain value usually.Show that thus effect and HGF that this 7-12SFd-F has free HGF from cell produce the promotion activity, the generation of HGF increases along with the increase of time.It remains on certain low value afterwards.This result is shown in table 46～48.

The HGF amount over time in table 46 culture medium

Incubation time (hour)

0????0.5????1?????2?????4?????8?????12????24????48????72

The HGF amount over time in the generation of HGF (ng/ hole) contrast 0.14 0.33 0.36 0.63 1.29 1.60 2.54 3.89 7.99 13.3 interpolation 7-0.48 1.82 2.16 2.53 3.12 4.01 5.84 9.82 17.0 23.512SFd-F table 47 cell

Incubation time (hour)

0?????0.5???1?????2?????4?????8?????12????24????48????72

The generation of HGF (ng/ hole) contrast 2.65 3.11 2.73 2.77 2.31 2.82 2.80 4.61 5.36 6.74 adds in 7-2.79 1.78 1.65 1.31 1.06 1.31 1.07 1.56 2.66 3.0912SFd-F table 48 culture medium and total HGF amount is over time in the cell

Incubation time (hour)

0?????0.5???1?????2?????4?????8?????12????24????48????72

7-3.26 3.60 3.81 3.84 4.18 5.31 6.91 11.4 19.7 26.612SFd-F are added in the generation of HGF (ng/ hole) contrast 2.79 3.43 3.09 3.39 3.60 4.41 5.34 8.50 13.3 20.2

(4) (CCL171: big Japanese pharmacy society system, code.02-021) 500 μ l add 48 porocyte culture plates, reach 1 * 10 will to be suspended in the MRC-5 cell of the DMEM culture medium that contains 10% hyclone ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DME culture medium exchange that contains 1% hyclone.Afterwards, add sample, cultivate further and cultivated 24 hours.Reclaim culture medium, (Funakoshi society system code.RS-0641-00), is measured the amount of HGF in the culture medium to use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit.Further, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mM HEPES pH7.4,10mM EDTA, 0.1%Triton X100,1mM PMSF, 1 μ/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.Adding 7-12SFd-F makes its ultimate density reach 1,10,100 μ g/ml.As negative control, add distilled water with sample equivalent.All tests are all carried out getting its meansigma methods 3 times.The result is shown in table 49.HGF concentration ratio negative control group in the culture medium of this 7-12SFd-F interpolation group significantly increases in the interdependent mode of 7-12SFd-F concentration.On the other hand, the intracellular HGF amount of this 7-12SFd-F interpolation group reduces in the interdependent mode of 7-12SFd-F concentration.Extracellular total HGF amount increases in the interdependent mode of concentration.Show that thus effect and HGF that this 7-12SFd-F has free HGF from cell produce the promotion activity.This result is shown in table 49.

The generation of HGF when table 49 adds 7-12SFd-F

7-12SFd-F addition (ultimate density μ g/ml)

0????????1?????????10???????100

6.28 6.04 4.15 1.59 total amounts 11.9 14.5 21.3 23.6 in 5.66 8.46 17.1 22.0 cells in the generation of HGF (ng/ hole) culture medium

Embodiment 6

(1) the MRC-5 Cell sap 500 μ l that will be suspended in the DMEM culture medium that contains 10% hyclone add 48 porocyte culture plates, reach 1 * 10 ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DME culture medium exchange that contains 1% hyclone.Afterwards, add cycloheximide (protein synthesis inhibitor: nacalaitesque society system), make it ultimate density and reach 0,1,10 μ g/ml, further add tested sample after, cultivated 24 hours.Reclaim culture medium, use HGF ELISA test kit, measure the amount of HGF in the culture medium.The fucoidan that adds the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record makes ultimate density reach 1,10,100 μ g/ml.In contrast, the distilled water of interpolation and sample equivalent.Test is all carried out getting its meansigma methods 2 times.The result is shown in table 50.In table 50, the HGF generation of matched group is decided to be 100%.

Shown in table 50, by adding cycloheximide, the HGF concentration in the fucoidan interpolation group culture medium reduces in the interdependent mode of cycloheximide concentration.Its suppression ratio suppresses in the interdependent mode of cycloheximide concentration, and the inhibition that causes with the cycloheximide of the matched group that does not add fucoidan has identical level.Hence one can see that, and it is synthetic to relate to albumen in the generation of the HGF that fucoidan causes is induced.

Fucoidan 01 10 (μ g/ml) the HGF generation of the addition (μ g/ml) of table 50 ガ go メ Thallus Laminariae (Thallus Eckloniae) origin cycloheximide and the ratio (%) of matched group contrast 100 35 27

1???????????????226????????110??????72

10??????????????317????????130??????110

100 456 206 127 (the HGF generation of matched group is 7.3ng/ml).

(2) (CCL171: big Japanese pharmacy society system, code.02-021) 500 μ l add 48 porocyte culture plates, reach 1 * 10 will to be suspended in the MRC-5 cell of the DME culture medium that contains 10% hyclone ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DMEM culture medium exchange that contains 1% hyclone.Afterwards, add cycloheximide (protein synthesis inhibitor: Na カ ラ ィ テ ス Network society system), make it ultimate density and reach 0,1,10 μ g/ml, further add tested sample after, cultivated 24 hours.Reclaim culture medium, (Funakoshi society system code.RS-0641-00), is measured the amount of HGF in the culture medium to use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit.Further, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mM HEPESpH7.4,10mM EDTA, 0.1%Triton X100,1mM PMSF, 1 μ/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.The generation of HGF, with negative control as 100%.Suppression ratio is that the HGF generation when only adding the 7-12SFd-F of each concentration is a benchmark, calculates the suppression ratio (%) that adds the cycloheximide component.Adding 7-12SFd-F makes its ultimate density reach 1,10,100 μ g/ml.As negative control, add distilled water with sample equivalent.All tests are all carried out getting its meansigma methods 3 times.Its result shown in table 51,52, by adding cycloheximide, in the culture medium of 7-12SFd-F interpolation group, in the cell and the amount of the total HGF in the culture medium all reduce in the interdependent mode of cycloheximide concentration.Showing that thus 7-12SFd-F induces the generation of HGF, is not the HGF that dissociates from cell simply, but synthetic relevant with albumen.Table 51 HGF produces addition (μ g/ml) dosage (μ g/ml) 01 10 that inductive inhibition (in the culture medium) 7-12SFd-F adds cycloheximide

HGF generation: %/conduct when not adding cycloheximide

100% suppression ratio: %

0??????????100/0??????40/60??????40/60

1??????????124/0??????61/51??????39/69

10?????????216/0??????88/59??????53/75

100 26,5/0 117,/56 74/72 table 52 HGF produce addition (μ g/ml) dosage (μ g/ml) 01 10 that inductive inhibition (adding up in the cell He in the culture medium) 7-12S Fd-F adds cycloheximide

HGF generation: %/conduct when not adding cycloheximide

100% suppression ratio: %

0??????????100/0??????31/69?????27/73

1??????????104/0??????31/70?????25/76

10?????????128/0??????38/70?????28/78

100????????137/0??????47/66?????37/73

(3) (CCL171: big Japanese pharmacy society system, code.02-021) 500 μ l add 48 porocyte culture plates, reach 1 * 10 will to be suspended in the MRC-5 cell of the DME culture medium that contains 10% hyclone ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DMEM culture medium exchange that contains 1% hyclone.Afterwards, add actinomycin D (rna synthesis inhibitor: Sigma society system), make it ultimate density and reach 0,0.1,1 μ g/ml, further add tested sample after, cultivated 24 hours.Reclaim culture medium, (Funakoshi society system code.RS-0641-00), is measured the amount of HGF in the culture medium to use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit.The generation of HGF, with negative control as 100%.Suppression ratio is that the HGF generation when only adding the fucoidan of each concentration is a benchmark, calculates the suppression ratio (%) that adds the actinomycin D component.The fucoidan that adds the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record makes its ultimate density reach 1,10,100 μ g/ml.As negative control, add distilled water with sample equivalent.All tests are all carried out getting its meansigma methods 2 times.Its result is shown in table 53, and by adding actinomycin D, the HGF concentration in the culture medium of this fucoidan interpolation group is suppressed in the interdependent mode of actinomycin D concentration.In the generation of the HGF that fucoidan causes is induced, there is the synthetic relevant probability with RNA in hint thus, shows not to be free HGF from cell simply.

Fucoidan 0 0.1 1 (μ g/ml) HGF generation: the %/do not do when adding actinomycin D of table 53 ガ go メ Thallus Laminariae (Thallus Eckloniae) origin actinomycin D addition (μ g/ml)

Be 100% suppression ratio: %

0??????????????100/0???????79/21?????83/17

1??????????????244/0???????138/43????119/51

10?????????????343/0???????224/35????239/30

100????????????492/0???????341/31????285/42

(4) (CCL171: big Japanese pharmacy society system, code.02-021) 500 μ l add 48 porocyte culture plates, reach 1 * 10 will to be suspended in the MRC-5 cell of the DME culture medium that contains 10% hyclone ⁵Cell/ml is at 37 ℃, 5%CO ₂Exist down and cultivate after 24 hours, with the DME culture medium exchange that contains 1% hyclone.Afterwards, add actinomycin D (rna synthesis inhibitor: Sigma society system), make it ultimate density and reach 0,0.1,1 μ g/ml, further add tested sample after, cultivated 24 hours.Reclaim culture medium, (Funakoshi society system code.RS-0641-00), is measured the amount of HGF in the culture medium to use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit.Further, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mM HEPES pH7.4,10mM EDTA, 0.1%Triton X100,1mM PMSF, 1 μ/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.The generation of HGF, with negative control as 100%.Suppression ratio is that the HGF generation when only adding the 7-12SFd-F of each concentration is a benchmark, calculates the suppression ratio (%) that adds the actinomycin D component.Adding 7-12SFd-F makes its ultimate density reach 1,10,100 μ g/ml.As negative control, add distilled water with sample equivalent.All tests are all carried out getting its meansigma methods 3 times.Its result shown in table 54,55, by adding actinomycin D, in the culture medium of 7-12SFd-F interpolation group, in the cell and the amount of the total HGF in the culture medium all reduce in the interdependent mode of actinomycin D concentration.In the generation of the HGF that 7-12SFd-F causes is induced, there is the synthetic relevant probability with RNA in hint thus, shows not to be free HGF from cell simply.

Table 54 HGF produces addition (μ g/ml) amount (the μ g/ml) 0 0.1 1 that inductive inhibition (in the culture medium) 7-12SFd-F adds actinomycin D

HGF generation: %/when not adding actinomycin D

Suppression ratio as 100%: %

0????????????100/0??????76/24??????80/20

1????????????130/0??????95/27??????96/26

10???????????225/0??????182/19?????152/32

100 29,5/0 212,/28 187/48 table 55 HGF produce addition (μ g/ml) amount (the μ g/ml) 0 0.1 1 that inductive inhibition (adding up in the cell He in the culture medium) 7-12SFd-F adds actinomycin D

HGF generation: %/conduct when not adding actinomycin D

100% suppression ratio: %

0??????????????100/0??????61/39???????59/41

1??????????????98/0???????64/35???????61/38

10?????????????113/0??????83/27???????71/37

100????????????126/0??????91/28???????81/36

Embodiment 7

(1) uses the male Wistar rat in 7 ages in week, by the following cut-out liver that carries out of Surgery Treatment.That is, under etherization, rat is cut open the belly, the root blood vessel is pricked the liver of back excision about 30% with the surgical stitch toe-in.The abdominal part that cuts is sewed up with sewing needle.

After the excision, use the fucoidan administration for the first time of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record at once, intraperitoneal administration in 12 hours at interval later on.In matched group, use the administration of normal saline intraperitoneal.

Behind the hepatectomy, at the 24th hour or 72 hours, the rat enlarged abdomen arterial blood drawing under the narcotism, the disodium EDTA with 0.1% is with the blood plasma centrifugalize.The HGF that measures in the blood plasma with HGFELISA test kit (Co., Ltd.'s specifc immunity institute) measures.

The result is shown in table 56.The number of elements of rat in representing every group in the numeral mean+/-standard error in the table, ().In addition, the * in the table represents to compare in the significant level below 5% with matched group evident difference.

Table 56

This medicine sample dosage plasma HGF concentration (ng/ml)

(mg/kg) 24 hours 72 hours

Normal saline 0.28 ± 0.04 (4) 0.30 ± 0.02 (3)

Rock 0.1 0.40 ± 0.12 (4) 0.52 ± 0.06 (5) * of (matched group) ガ go メ Thallus Laminariae (Thallus Eckloniae) origin

Algae is according to polysaccharide 1 0.65 ± 0.18 (4) 0.64 ± 0.09 (2) *

Fucoidan administration group is compared with matched group, can be observed the tendency that the plasma HGF amount rises after behind the hepatectomy 24 hours, can be observed tangible rising after 72 hours.

From as can be known above, because the fucoidan generation of inducing HGF, it is useful therefore promoting liver to regenerate rapidly and recover in the remaining liver function at the postoperative of the operating hepatopathy of needs.

(2) use the male Wistar rat in 7 ages in week, by Surgery Treatment cut-out liver.Under etherization cut open the belly, the root blood vessel is pricked the liver of back excision about 30% with the surgical stitch toe-in.The abdominal part that cuts is sewed up with sewing needle.After the excision, the F-fucoidan administration for the first time that the enzyme that uses reference example 2-(4) to prepare is at once handled separates oral administration twice later on sooner or later.In matched group, use the normal saline administration.Behind the hepatectomy, at the rat enlarged abdomen arterial blood drawing under the narcotism after the 24th hour, the disodium EDTA with 0.1% is with the blood plasma centrifugalize.The HGF that measures in the blood plasma with HGF ELISA test kit (Co., Ltd.'s specifc immunity institute) measures.

The result is shown in table 57.The number of elements of rat in representing every group in the numeral mean+/-standard error in the table, ().In addition, the * in the table represents to compare in the significant level below 5% with matched group evident difference.

Table 57

Plasma HGF concentration (ng/ml)

Organize 24 hours

F-fucoidan 0.505 ± 0.97* (5) that normal saline 0.184 ± 0.33 (6) enzyme is handled

(1g/kg/ days)

The F-fucoidan administration group that enzyme is handled is compared with matched group, can be observed tangible rising after behind the hepatectomy 24 hours.

From as can be known above, owing to contain the generation that the F-fucoidan of 7-12SFd-F is induced HGF in a large number, it is useful therefore promoting liver to regenerate rapidly and recover in the remaining liver function at the postoperative of the operating hepatopathy of needs.

Embodiment 8

To highly express people's neonate foreskin epithelial cell strain Hs68 cell (ATCC CRL-1635) as a kind of h-IGF-1 of Insulin-Like multiplicaiton factor in the DMEM culture medium that contains 10% hyclone (FBS:Bio Whittaker society system) (Gibco BRL society system), in the presence of 5%CO2,37 ℃ be cultured to cell in incubator saturated till, make it reach 3 * 10 in above-mentioned culture medium cell suspension with trypsin-EDTA solution (Bio Whittaker society system) ³Individual/hole, in each hole of 96 hole microdroplet plates, add 200 μ l respectively.Cultivate after 5～7 days, almost incubator is removed culture medium when saturated at cell, in every hole, added the above-mentioned culture medium 200 μ l of the fucoidan of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of record among component I, component I I, component III or the reference example 1-(1) of record among the reference example 1-(2) that contains 0,12.3,37,111,333,1000 or 3000 μ g/ml respectively.With 24 hours time sampling, reclaim culture supernatant through 1,4,12,24 hour, the H-IGF that measures for the Hs68 cell with h-IGF-1ELISA test kit (Diagnostique society system) produces induced activity.The result is shown in table 58～61.In addition, do not add sample in contrast.

Fucose (ng/ml) (μ g/ml) contrast 4.3 2.9 4.1 4.5 12.3 22.9 14.7 10.5 11.8 37 17.5 13.9 10.8 11.4 111 14.6 13.7 10.3 7.8 333 13.8 17.6 9.4 7.7 1,000 13.9 14.7 14.6 7.5 3,000 15.9 14.0 14.0 7.2 in 1 hour 4 hours 12 hours 24 hours of h-IGF-1 concentration in the table 58 ガ go メ kelp origin culture medium

H-IGF-1 concentration in the table 59 component I culture medium (μ g/ml) (ng/ml)

Contrast 6.8 5.4 5.0 5.0 12.3 13.9 10.5 7.6 7.3 37 19.0 11.7 8.3 10.1 111 20.4 11.0 9.5 9.9 333 18.7 12.2 10.9 10.3 1,000 17.7 13.2 12.7 11.3 3,000 19.0 12.1 13.1 11.7 in 1 hour 4 hours 12 hours 24 hours

H-IGF-1 concentration (μ g/ml) (ng/ml) in the table 60 component I I culture medium

Contrast 4.3 2.9 4.1 4.5 12.3 18.5 10.2 6.9 7.0 37 20.1 9.9 9.2 9.2 111 20.0 11.1 9.8 8.9 333 16.3 12.7 9.7 9.2 1,000 17.0 12.2 10.0 8.8 3,000 17.9 11.3 10.0 7.6 in 1 hour 4 hours 12 hours 24 hours

H-IGF-1 concentration in the table 61 component III culture medium (μ g/ml) (ng/ml)

Contrast 4.3 2.9 4.1 4.5 12.3 16.5 10.9 8.5 8.6 37 16.7 in 1 hour 4 hours 12 hours 24 hours. 10.2 11.1 8.4 111 11.8 11.5 8.6 6.9 333 9.6 11.2 8.0 5.9 1,000 7.9 10.4 10.6 6.6 3,000 7.1 9.4 9.7 6.2

Shown in table 58～61, the fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin, component I, component I I and component III show that h-IGF-1 produces induced activity.By adding the sample of 12～100 μ g/ml, h-IGF-1 produces induced activity and showed peak at the 1st hour.In addition, do not find toxicity, the proliferation inhibition activity of each sample to the Hs68 cell.For other acidic polysaccharose of putting down in writing in the reference example, its degradation product, acidic oligosaccharide, acid monosaccharide and their salt, can confirm to have h-IGF-1 to produce induced activity too.

Embodiment 9

M199 culture medium (ICN society system) with rat becomes fiber L-M cell (ATCC CCL-1.2) to be suspended in to contain 0.5%bactopepton (Difco society system) reaches 1.5 * 10 ⁵Cell/ml adds 96 orifice plates with it, and every hole 0.1ml carries out aseptic culture.

Cultivate after 3 days, remove culture medium, with the M199 culture medium displacement that contains 0.5% bovine serum albumin (Sigma society system).The fucoidan of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of record reaches concentration 0,62.5,250,1000 μ g/ml in wherein adding reference example 1-(1), cultivates 24 hours.Use the material that adds distilled water in contrast.After cultivation is finished, with the NGF concentration in enzyme immunoassay (EIA) (NGF Emax Immuno Assay System:Promega society system) the mensuration culture fluid.With the contrast the NGF generation as 100%, all tests are carried out 2 times and are averaged.The results are shown in table 62.Shown in table 62, the fucoidan of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin promotes the generation of L-M cellular neural multiplicaiton factor in the interdependent mode of concentration.In addition, its fractional distillation component also shows same activity.In addition, other acidic polysaccharose of putting down in writing in the reference example, its degradation product, acidic oligosaccharide, acid monosaccharide and their salt show that too NGF produces induced activity.

The increase of table 62 sample solution concentration NGF generation

(μ g/ml) be the fucoidan 250 166 of ガ go メ Thallus Laminariae (Thallus Eckloniae) origin 62.5 117 (%)

1,000 179 (the NGF generation of matched group is 155pg/ml).

Measure the activity of the neural multiplicaiton factor generation of promotion of the middle component I of putting down in writing of reference example 1-(2), component I I, component III in the same way, confirm that each component has activity.Its result is shown in table 63.In addition, other acidic polysaccharose of putting down in writing in the reference example, its degradation product, acidic oligosaccharide, acid monosaccharide and their salt show that too NGF produces inducing action.

Table 63

The increase of sample solution concentration NGF generation

(μg/ml)?????????(％)

Component I 250 505.6

500????????????619.6

1000???????????806.5

Component I I 250 664.5

500????????????864.5

1000???????????1137.4

Component III 250 1021.1

500????????????1187.0

1,000 1265.0 (the NGF generation of matched group is 50.03pg/ml).

Embodiment 10

(1) buy male C3H/He mice from Japanese SLC society, preparation is raised the back and is tested with the mice in 5 all ages.The fucoidan of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) preparation is reached 3% concentration with the ethanol dissolving that suspends, to the back coated 200 μ l of every mice.Matched group carries out same coating with ethanol.Administration 1 day 1 time was carried out 8 days continuously.Begin in administration the 9th day,, measure HGF activity in the skin with ELISA test kit (Co., Ltd.'s specifc immunity institute) with skin peeling.

The result is shown in table 64.Numeral in the table be the mean+/-standard error of 5 examples.

The HGF specific activity matched group that extracts from skin in fucoidan coating group has tangible rising, and can confirm to be coated with fucoidan has HGF to produce inducing action.

Table 64

HGF activity in the skin

(ng/g tissue)

Matched group (N=5) 16.31 ± 2.86 fucoidan coating groups (N=5) 104.46 ± 4.05 meansigma methodss ± standard deviation

(2) aftermentioned embodiment 26-(1) record is compared astringent of the present invention and the contrast astringent that do not contain fucoidan, 20～35 years old adult women, 25 people are carried out functional check under their unwitting situation.Its result, effectively result and judgement number are shown in table 65.

Table 65

The flexible astringent 21 19 16 contrast astringent 569 of the present invention of skin wet skin smooth skin

From above result as can be known, the HGF by fucoidan produces inducing action, and the astringent of the present invention that has mixed fucoidan is all demonstrating outstanding effect aspect moistening, smooth, the elasticity of skin.

Embodiment 11

(1) the F-fucoidan 98mg that will prepare with the method for reference example 1-(2) record is dissolved among the DMS0 5ml, after room temperature adds 980mg piperidines sulphuric acid, stirs 2 hours at 80 ℃.After the reactant liquor cooling, with the dialyzer dialysis of getting rid of molecular weight 1,000 2 days.The gained dialyzed solution joins cation exchange column [Amberlite IRA-120 (Na ⁺)] on, prepare the high hydrosulphate of F-fucoidan by drying under reduced pressure.

(2) the 7-12SFd-F 34mg that will prepare according to the method for reference example 2 records is dissolved among the DMS0 4ml, afterwards, according to the same operation of embodiment 11-(1), the high hydrosulphate of preparation 7-12SFd-F.

(3) according to the same quadrat method of embodiment 1, the HGF generation of the 7-12SFd-F (sample 4.) of the high hydrosulphate (sample 2.) of the 7-12SFd-F of the high hydrosulphate (sample 1.) of the F-fucoidan of mensuration embodiment 11-(1) preparation, embodiment 11-(2) preparation, the F-fucoidan (sample 3.) of reference example 1-(2) preparation and reference example 2 preparations is active.Adding each sample makes its ultimate density reach 1,10,100 μ g/ml respectively.In contrast, the distilled water of interpolation and sample equivalent.

Its result shown in table 66, in table 66, with the contrast the HGF volume of production as 100%.All tests are all carried out getting its meansigma methods 2 times.Shown in table 66,1.～4. sample all induces the generation of HGF.In addition, the HGF of high hydrosulphate produces the not material height of high-sulfur acidification of induced activity ratio.Hence one can see that, can strengthen its HGF generation induced activity by already present natural sulphur acidify sugar further being carried out the sulphation processing.

In addition, (1～10mg/ml) solution was 105 ℃ of heating 4 hours by the 1N HCl 0.2ml with each sample, in 0.1ml, add 0.1N HCl solution 1.9ml and 1% barium chloride-0.5% gelatin solution 0.25ml afterwards, place after 20 minutes, measure absorbance at 500nm and carry out the quantitative of sulfuric acid content.Inspection amount line be with 0,1,3,5,7,10,15, the 1N HCl solution of 10mM sodium sulfate makes as standard specimen, the sulfuric acid content of obtaining each sample from these inspection amount lines (is scaled SO ₃).This inspection amount line as shown in Figure 2, the sulfuric acid content of each sample is shown in table 67.

Generation (%) (μ g/ml) sample of table 66 concentration HGF is 2. 3. 4. 1 346 192 312 192 10 715 214 493 229 100 794 499 598 355 (the HGF generation of matched group is 8.15ng/ml) of sample of sample of sample 1..

Table 67

The sample sulfuric acid content (is scaled SO ₃, %)

The high hydrosulphate 47 of F-fucoidan 40F-fucoidan

The high hydrosulphate 48 of 7-12SFd-F 407-12SFd-F

Embodiment 12

Will be at NHDF cell (people's normal skin fibroblast: Bio Wittaker society system) be suspended in the DMEM culture medium that contains 10% hyclone, reach 1 * 10 of the DMEM culture medium culturing that contains 10% hyclone ⁵Cell/ml adds 48 porocyte culture plates with it, and every hole 500 μ l cultivated 24 hours.Afterwards, with the DMEM culture medium exchange that contains 1% hyclone, afterwards, add myristoyl phorbol 13-acetate (TPA:Gibco BRL society system) and the sample of 10nM.Cultivated again 20 hours, and reclaimed culture medium, use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit (Funakoshi society system, Code.RS-0641-00), measure the HGF amount in the culture medium.The HGF generation with negative control as 100%.The ultimate density of each sample is respectively: the fucoidan that adds reference example 1-(1) record reaches 1,10,100 μ g/ml, adds heparin and reaches 1,10 μ g/ml.As negative control, add distilled water with sample equivalent.The cultivation of induction experiment, the TPA of interpolation 10nM when adding sample.Test is all carried out 3 times, gets its meansigma methods.The result is shown in table 68.All groups of cells of adding the fucoidan sample are significantly increased than the HGF generation of the matched group that adds distilled water.In addition, compare with adding heparin, the generation of HGF also significantly increases.Hence one can see that, and the fucoidan of reference example 1-(1) record has the heparin of HGF inducing action to show that higher HGF generation promotes active than up to the present confirming.

Table 68 concentration heparin fucoidan (μ g/ml)

0?????????100?????????100

1?????????950?????????1140

10????????2170????????2470

100-2770 (condition is that the HGF generation of matched group is 0.20ng/ml).

Embodiment 13

To be suspended in the DMEM culture medium that contains 10% hyclone at the Hs68 cell of the DMEM culture medium culturing that contains 10% hyclone (people's neonate foreskin cell: big Japanese pharmacy society system, ATCC CRL-1635), reach 1 * 10 ⁵Cell/ml adds 48 porocyte culture plates with it, and every hole 500 μ l cultivated 24 hours.Afterwards, with the DMEM culture medium exchange that contains 1% hyclone, add TPA and the sample of 10nM.In addition, do not add TPA, only add sample and carry out equally as distinguishing.Reclaim culture medium, use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit, measure the amount of HGF in the culture medium.Further, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mMHEPES pH7.4,10mM EDTA, 0.1%Triton X100,1mM PMSF, 1 μ/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.Add the 7-12SFd-F of reference example 2-(3) preparation, make its ultimate density reach 0.1,1,10,100 μ g/ml.As negative control, add distilled water with sample equivalent.All tests are all carried out getting its meansigma methods 3 times.Its result is shown in table 69～71.Not observing significant HGF under the situation of not adding TPA produces.But when adding TPA, the HGF amount in the cell reduces in the interdependent mode of concentration of 7-12SFd-F, and the HGF amount in the culture medium and total HGF amount increase in the interdependent mode of concentration of 7-12SFd-F.In addition, total HGF amount has been compared tangible increase with the matched group that does not add TPA.In addition, the increase of HGF, the mRNA situation of handling with no TPA under the situation that such mRNA increases is seldom compared, and the increase of highly significant is arranged.Hence one can see that, and 7-12SFd-F promotes transcribing of mRNA, when a large amount of HGF of needs, can significantly promote the free of HGF and produce.HGF amount (pg/ hole) in the table 69 Hs68/7-12SFd-F culture medium

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0???????????????N.D.??????????65.54

0.1?????????????N.D.??????????71.04

1???????????????N.D.??????????99.25

10??????????????N.D.??????????226.14

100 N.D. 260.49N.D. are illustrated in below the detection limit.HGF amount (pg/ hole) in the table 70 Hs68/7-12SFd-F cell

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0????????????98.62???????????155.65

0.1??????????87.60???????????151.99

1????????????62.47???????????142.17

10???????????N.D.????????????120.70

100 N.D. 95.67N.D. are illustrated in below the detection limit.The total HGF amount of table 71 Hs68/7-12SFd-F (pg/ hole) 7-12SFd-F does not have TPA 10nMTPA (μ g/ml)

0???????????????????????221.19

0.1?????????????????????223.02

1???????????????????????241.42

10??????????????????????346.84

100?????????????????????356.05

Embodiment 14

Will the MRC-5 cell of the DMEM culture medium culturing that contains 10% hyclone (CCL171: big Japanese pharmacy society system, code.02-021) be suspended in the DMEM culture medium that contains 10% hyclone, reach 2.5 * 10 ⁵Cell/ml adds 6 porocyte culture plates with it, in the presence of 5% carbon dioxide, cultivates 24 hours at 37 ℃.Afterwards, with the DMEM culture medium exchange that contains 1% hyclone, cultivated again 22 hours.Then, reach the culture fluid of 100 μ g/ml respectively with the 7-12SFd-F ultimate density of having added reference example 2-(3) preparation, add the culture fluid that LM heparin (Celsus laboratories society system) reaches 1 μ g/ml, add the prostaglandin E that is dissolved in the dimethyl sulfoxide (DMSO) ₁(PGE ₁) (with the pure pharmaceutical worker of light industry society system) culture fluid of reaching 1 μ M exchanges.In contrast, use the culture medium of having added DMSO.In addition, the solvent of above-mentioned each additive of interpolation makes them all reach 1%.Further cultivate, extracted whole RNA at the 0th, 2,4,6,8,10,12,24 hour.When the whole RNA of extraction, use Rneasy Mini test kit (QIAGEN) society system.RT-PCR uses RNA PCR test kit ver.2.1 (precious wine is made the system R019A of society).After carrying out full RNA thermal denaturation processing, use random primer (N6) (precious wine is made society's system, 3801) to carry out 10 minutes at 30 ℃, 42 ℃ were carried out 30 minutes, and 99 ℃ are carried out 5 minutes reverse transcription reactions.For detecting the mRNA of HGF,, use the primer of putting down in writing in the sequence table sequence sequence number 2 as antisense primer as there being adopted primer to use the primer of record in the sequence table sequence sequence number 1.Product by this primer amplification is 415bp.In addition, for carrying out semiquantitative test, also carry out detection as the β actin of house-keeping gene.As there being adopted primer to use the primer of record in the sequence table sequence sequence number 3, use the primer of putting down in writing in the sequence table sequence sequence number 4 as antisense primer.Product by this primer amplification is 275bp.PCR is undertaken by PJ 9600 (Perkin-Elmer corporate system).PCR circulate in 94 ℃ carry out 2 minutes thermal denaturations after, will carry out 30 seconds thermal denaturations at 94 ℃, carry out annealing in 30 seconds at 59 ℃, carry out 24 times 72 ℃ of circulations of carrying out 60 seconds extensions.After the reaction, carry out 2% agarose gel electrophoresis and ethidium bromide staining, under the UV irradiation, observe gel.In all samples, all detected the mRNA of HGF.In contrast, can be observed PGE ₁Inductive mRNA, but do not observe 7-12SFd-F and LM heparin inducing to mRNA.Hence one can see that, in the state that the mRNA of common HGF is transcribed, can not promote the transcribing of mRNA of HGF by adding 7-12SFd-F and LM heparin, and therefore as can be known, 7-12SFd-F and LM heparin can not cause that superfluous HGF produces and induce.

Embodiment 15

(people's normal skin fibroblast: Bio Whittaker society system) the HS68 cell that uses among the replacement embodiment 13, other carry out similarly to Example 13, study 7-12SFd-F generation induced activity to HGF in the NHDF cell with the NHDF cell.Its result is shown in table 72～74.

When not adding TPA, HGF amount in the cell reduces in the interdependent mode of 7-12SFd-F concentration, HGF amount in the culture medium and total HGF amount increase in the interdependent mode of 7-12SFd-F concentration, and total HGF amount has been compared tangible increase with the matched group that does not add 7-12SFd-F.Hence one can see that, even under the few situation of the mRNA that resembles the no TPA HGF handling, also can promote from synthetic two aspects of the free and HGF of the HGF of cell surface.In addition, when adding TPA, the HGF amount in the cell reduces in the interdependent mode of 7-12SFd-F concentration, and the HGF amount in the culture medium and total HGF amount increase in the interdependent mode of 7-12SFd-F concentration, and total HGF amount is compared with the matched group that does not add 7-12SFd-F and is significantly increased.In addition, like this when mRNA increases, mRNA situation is seldom compared the increase highly significant of HGF when not adding TPA.Hence one can see that, and 7-12SFd-F is under a spot of situation at mRNA, promotes the free and generation of HGF to a certain extent, and, promoted transcribing of mRNA, when needing a large amount of HGF, can significantly promote the free and generation of HGF.The amount of HGF (ng/ hole) in the table 72 NHDF/7-12SFd-F culture medium

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0?????????N.D.???????0.39

1?????????0.05???????0.68

10????????0.18???????1.22

100 0.385 1.625N.D. represent below the detection limit.The amount of HGF (ng/ hole) in the table 73 NHDF/7-12SFd-F cell

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0?????????0.185?????0.24

1?????????0.145?????0.19

10????????0.075?????0.12

The amount (ng/ hole) of the total HGF of 100 0.025 0.055 table 74 NHDF/7-12SFd-F

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0???????????????????0.63

1????????0.195??????0.87

10???????0.255??????1.335

100??????0.41???????1.68

Embodiment 16

To be suspended in the DMEM culture medium that contains 10% hyclone at the NHDF cell (people's normal skin fibroblast) of the DMEM culture medium culturing that contains 10% hyclone, reach 2.5 * 10 ⁵Cell/ml, every hole 2ml adds 6 porocyte culture plates with it, in the presence of 5% carbon dioxide, cultivates 24 hours at 37 ℃.Afterwards,, add the myristoyl phorbol 13-acetate (TPA:Gibco BRL society system) of 10nM and the 7-12SFd-F of reference example 2-(3) preparation, make ultimate density reach 100 μ g/ml with the DMEM culture medium exchange that contains 1% hyclone.In addition,, do not add TPA, only add 7-12SFd-F, carry out equally as difference.Further cultivate, extracted whole RNA at the 4th, 6,8,10 hour.When the whole RNA of extraction, use Rneasy Mini test kit (QIAGEN society system).RT-PCR except PCR circulation is carried out 28 times with embodiment 14 identical carrying out.After the reaction, carry out 2% agarose gel electrophoresis and ethidium bromide staining, under the UV irradiation, observe gel.

Its result, when not adding TPA, the amount of transcribing of the mRNA of HGF is considerably less, but by adding 7-12SFd-F, adds back 4 hours, can be observed the rising that a spot of mRNA transcribes.On the other hand, by adding TPA, when the mRNA amount of HGF is no matter all significantly rise.When adding TPA and 7-12SFd-F, compare with the situation of only adding TPA, the mRNA amount of HGF was increased in interpolation in back 4 hours, but in the time of 6 hours, with interpolation 7-12SFd-F indifference.That is, at the initial stage that transcribing of the mRNA that needs HGF is activated, 7-12SFd-F can significantly promote it to transcribe, but after, its facilitation effect disappears.Hence one can see that, the generation that 7-12SFd-F induces HGF in the moment that needs HGF, afterwards, the not generation that can superfluous induce HGF.

Embodiment 17

To be suspended in RPMI 1640 culture medium that contain 1% hyclone at the HL60 cell (people's prebone marrow leukaemia cell) of RPMI 1640 culture medium culturings that contain 10% hyclone, reach 5 * 10 ⁵Cell/ml adds 48 porocyte culture plates with it, every hole 500 μ l.Afterwards, add the TPA of 10nM, further add sample, cultivated 24 hours.In addition, do not add TPA, only add sample and carry out equally as distinguishing.Reclaim culture medium, use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit, measure the amount of HGF in the culture medium.Further, after cell cleaned with PBS, it is dissolved in the cytolysis buffer (50mM HEPES pH7.4,10mM EDTA, 0.1%TritonX100,1mM PMSF, 1 μ/ml pepsin inhibitor A, 1 μ g/ml leupeptin) of 500 μ l.In order to make it dissolve available ultrasonic Treatment fully, centrifugalize afterwards prepares supernatant (cell extraction liquid), uses and measures in the culture medium the same quadrat method of HGF concentration and measure intracellular HGF amount.Add the 7-12SFd-F of reference example 2-(3) preparation, make its ultimate density reach 1,10,100 μ g/ml.As negative control, add distilled water with sample equivalent.All tests are all carried out getting its meansigma methods 3 times.Its result is shown in table 75～77.

Under the situation of not adding TPA, HGF in cell amount is not with the concentration change of 7-12SFd-F.HGF amount in the culture medium can be observed increase at 1001 μ g/ml, but changes with the concentration of 7-12SFd-F is not significant.In addition, when adding TPA, though the amount of the HGF in the cell is overall in lower value not with the concentration change of 7-12SFd-F.On the other hand, the amount of the HGF in the culture medium and total HGF amount significantly increase in the interdependent mode of concentration of 7-12SFd-F.In addition, total HGF amount has been compared tangible increase with the matched group that does not add 7-12SFd-F.In addition, the increase of HGF, the mRNA situation of handling with no TPA under the situation that such mRNA increases is seldom compared, and the increase of highly significant is arranged.Hence one can see that, and 7-12SFd-F promotes transcribing of mRNA, when a large amount of HGF of needs, can significantly promote the free of HGF and produce.The amount of HGF (ng/ hole) in the table 75 HL60/7-12SFd-F culture medium

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0?????????76.74?????261.60

1?????????67.10?????295.94

10????????78.54?????464.55

The amount (ng/ hole) of HGF in the 100 162.85 843.84 table 76 HL60/7-12SFd-F cells

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0?????????294.12?????83.37

1?????????276.67?????89.39

10????????236.30?????67.09

The amount (ng/ hole) of the total HGF of 100 257.04 85.77 table 77 HL60/7-12SFd-F

7-12SFd-F does not have TPA 10nMTPA

(μg/ml)

0????????370.85?????344.97

1????????343.77?????385.32

10???????314.84?????531.64

100??????420.26?????929.60

Embodiment 18

To be suspended in RPMI 1640 culture medium that contain 1% hyclone at the HL60 cell (people's prebone marrow leukaemia cell) of RPMI 1640 culture medium culturings that contain 10% hyclone, reach 5 * 10 ⁵Cell/ml adds 6 porocyte culture plates with it, after every hole 2ml., adds the 7-12SFd-F of 10nMTPA and reference example 2-(3) preparation, makes ultimate density reach 100 μ g/ml.In addition,, do not add TPA, only add 7-12SFd-F, carry out equally as difference.Further cultivate, extracted whole RNA at the 4th, 6,8,10 hour.When the whole RNA of extraction, use Rneasy Mini test kit (QIAGEN) society system.RT-PCR except PCR circulation is carried out 32 times with embodiment 14 identical carrying out.After the reaction, carry out 2% agarose gel electrophoresis and ethidium bromide staining, under the UV irradiation, observe gel.

Its result, when not adding TPA, the amount of transcribing of the mRNA of HGF is considerably less, but by adding 7-12SFd-F, adds back 4 hours, can be observed the rising that the mRNA of a spot of HGF transcribes.On the other hand, by adding TPA, when the mRNA amount of HGF is no matter all significantly rise.When adding TPA and 7-12SFd-F, compare with the situation of only adding TPA, the mRNA amount of HGF was increased in interpolation in back 4 hours, but in the time of 6 hours, with interpolation 7-12SFd-F indifference.That is, at the initial stage that transcribing of the mRNA that needs HGF is activated, 7-12SFd-F can significantly promote it to transcribe, but after, its facilitation effect disappears.Hence one can see that, the generation that 7-12SFd-F induces HGF in the moment that needs HGF, afterwards, the not generation that can superfluous induce HGF.

Embodiment 19

(1) commercially available Caulis et Folium Chrysanthemi segeti lyophilization is obtained the lyophilization thing of Caulis et Folium Chrysanthemi segeti.This Caulis et Folium Chrysanthemi segeti lyophilization thing is pulverized, and gained Caulis et Folium Chrysanthemi segeti powder 10g is suspended in the 100ml chloroform, and the operation of filtered and recycled indissolvable component is repeated 3 times.Afterwards, be suspended in the 100ml ethanol and filter, the operation of reclaiming indissolvable component repeats 3 times.From this insoluble matter, remove ethanol fully, it is suspended in the 100ml distilled water.This suspension, is filtered after 1 hour 60 ℃ of insulations.In filtrate, add the ethanol of 2.5 times of amounts, after-20 ℃ of coolings, obtain precipitation in the low-temperature centrifugation separation.In distilled water, lyophilization obtains containing component, the Caulis et Folium Chrysanthemi segeti extract of Powdered sugar with this resolution of precipitate.

(2) according to the same quadrat method of embodiment 1-(1), the HGF that measures the Caulis et Folium Chrysanthemi segeti extract of embodiment 19-(1) preparation produces induced activity.Adding sample makes its ultimate density reach 1,10,100 μ g/ml.As negative control, make an addition to the distilled water of sample equivalent.The generation of HGF is as 100% with negative control.Its result is shown in table 78.All experiments are all carried out getting its meansigma methods 2 times.Shown in table 78, the Caulis et Folium Chrysanthemi segeti extract is induced the generation of HGF.

The generation (%) of table 78 Caulis et Folium Chrysanthemi segeti extract (μ g/ml) HGF

1??????????????????125

10?????????????????148

100 314 (condition is that the HGF generation of matched group is 8.21ng/ml.)

Embodiment 20

According to the same quadrat method of embodiment 1-(1), the HGF that measures the Radix Artemisia ordosicae supernatant component of reference example 13-(5) preparation produces induced activity.Condition is to add Radix Artemisia ordosicae supernatant component with the amount of culture medium 1/1000th.Its result is shown in table 79.Its result shows, the Radix Artemisia ordosicae extract promptly uses ethanol to precipitate and non-setting component also has HGF and produces induced activity.Can think thus, precipitate and non-setting lower-molecular-weight component also has activity with ethanol.

Table 79

The generation of Radix Artemisia ordosicae supernatant component HGF

Addition (%)

0???????????????????????100

1 463/1000ths (condition is that the HGF generation of matched group is 4.31ng/ml.)

Embodiment 21

(1) (sell the merchant: this Chinese prescription of slope hall) 50g puts into homogenizer (Japanese smart machine society system), adds 500ml acetone, 8000rpm homogenize 10 minutes, uses filter paper filtering, obtains residue with dry Folium Artemisiae Argyi.Repeat above operation 2 times, the Folium Artemisiae Argyi residue of gained 100g is put into homogenizer, add 500ml acetone,, use filter paper filtering, obtain residue 8000rpm homogenize 10 minutes.Repeat this operation 4 times, obtain the residue of cleaning with acetone.With acetone residue of cleaning and the washing with alcohol of cleaning identical usefulness 90% with acetone 4 times, 80% washing with alcohol 4 times obtains the clean residue of ethanol.From beginning most to repeat this operation 1 time, merge the ethanol that obtains the 200g Folium Artemisiae Argyi and clean residue.

(2) in the residue that ethanol is cleaned, add the sodium chloride of 10 liters of 100mM and contain 10% alcoholic acid 30mM phosphate buffer (pH8.0),, use filter paper filtering, obtain thick extract (filtrate) stirring at room 19 hours.After the thick extract of gained is concentrated into 2 liters with the ultrafiltration apparatus that is equipped with the hollow fibre of getting rid of molecular weight 10,000, adds 20 liters and carry out ultrafiltration when containing 10% alcoholic acid 100mM sodium chloride.Afterwards, be concentrated into 668ml, add the 1g active carbon, after 30 minutes,, remove active carbon 10000rpm centrifugalize 40 minutes in stirring at room.Trace active charcoal residual in the supernatant is removed with the No.5c filter.Take out 66.8ml from charcoal treatment liquid, after fully dialysing with distilled water, lyophilization obtains the dry thing of 670mg.Should drying thing called after Radix Artemisia ordosicae high molecular component (YPS).Residual 601.2ml charcoal treatment liquid is put into ultrafiltration apparatus,, obtain the Radix Artemisia ordosicae high molecular component of solvent exchange with containing 10% ethanol and 50mM sodium chloride 10mM imidazoles-hydrochloride buffer (pH7.0) carries out solvent exchange.

(3) the Radix Artemisia ordosicae high molecular component with solvent exchange is added to the DEAE-Cellulofine A-800 post of 10mM imidazoles-hydrochloride buffer (pH7.0) equilibrating that is contained 10% ethanol and 50mM sodium chloride (on the φ 4.05 * 37.8cm), after with same buffer 1200ml pillar being cleaned, carry out gradient elution to the sodium chloride of 2M (1000ml) with 0.1M (1000ml).Eluent is with every bottle of 30ml fractional distillation.In the component that eluting goes out, respectively with fraction No.13～33 called after Radix Artemisia ordosicae high molecular component-I (YPS-I), fraction No.69～78 called after Radix Artemisia ordosicae high molecular component-II (YPS-II), fraction No.79～137 called after Radix Artemisia ordosicae high molecular component-III (YPS-III).YPS-I, YPS-II, YPS-III are fully dialysed with distilled water respectively, and lyophilization obtains lyophilized products 530mg, 420mg, 380mg respectively.

(4) the further fractional distillation of YPS-III, YPS-III (200mg) is dissolved in 50ml to be contained in the 5mM imidazoles-hydrochloride buffer (pH8.0) of 4M sodium chloride, add it to same buffer equilibrating phenyl-Cellulofine post (on the φ 3.1 * 14.3cm), after with same buffer 200ml pillar being cleaned, 5mM imidazoles-the hydrochloride buffer (pH8.0) that contains 1M sodium chloride respectively with 200ml, the 200ml distilled water, 200ml ethanol carries out eluting.

Eluent is with every bottle of 10ml fractional distillation.In the component that eluting goes out, respectively with fraction No.1～32 called after Radix Artemisia ordosicae high molecular component-III-1 (YPS-III-1), fraction No.33～53 called after Radix Artemisia ordosicae high molecular component-III-2 (YPS-III-2), fraction No.54～66 called after Radix Artemisia ordosicae high molecular component-III-3 (YPS-III-3).YPS-III-1, YPS-III-2, YPS-III-3 are fully dialysed with distilled water respectively, and lyophilization obtains lyophilized products 20.11mg, 32.59mg, 113.75mg respectively.

(5) according to the same quadrat method of embodiment 1-(1) enzyme, measure embodiment 21-(2), the fraction of the Radix Artemisia ordosicae extract of 21-(3) preparation, the HGF of YPS (sample 1.), YPS-I (sample 2.), YPS-II (sample 3.), YPS-III (sample 4.) produces induced activity.The result is shown in table 80.Its result, the fraction of these Radix Artemisia ordosicae extracts shows that all HGF produces induced activity.

Table 80

The generation of sample solution concentration HGF

(μ g/ml) be sample 1. 0 100 (%)

1???????????214

10??????????267

100 215 samples 2. 0 100

1???????????121

10??????????113

100 260 samples 3. 0 100

10??????????129

100 243 samples 4. 0 100

1???????????232

10??????????323

100 272 (condition is that the HGF generation of matched group is 8.43ng/ml.)

(6) according to the same quadrat method of embodiment 1-(1), measure the fraction of the Radix Artemisia ordosicae extract of embodiment 21-(4) preparation, the HGF of YPS-III-1 (sample is 1.), YPS-III-2 (sample is 2.), YPS-III-3 (sample is 3.) produces induced activity.The result is shown in table 81.Its result, the fraction of these Radix Artemisia ordosicae extracts shows that all HGF produces induced activity.

Generation (%) (μ g/ml) sample of table 81 addition HGF 1. sample 2. sample 3. 0 100 100 100 1 188 290 247 10 181 304 217 100 348 295 243 (condition is that the HGF generation of matched group is 8.26ng/ml.)

Embodiment 22

Fucoidan 30g stirring at room in the 12L distilled water of the ガ go メ Thallus Laminariae (Thallus Eckloniae) origin of reference example 1-(1) record was dissolved in 30 minutes.This suspension 10000 * g centrifugalize 40 minutes, is collected supernatant.It with membrane filter (0.22 μ m) (Miuipore society system) aseptic filtration, is obtained 21.4g lyophilization thing.Be Takara fucoidan Bf (hereinafter referred to as fucoidan Bf).

With the same quadrat method of embodiment 1-(1), the HGF that measures fucoidan Bf (sample 1.) produces induced activity.Its result is shown in table 82.Experiment is carried out getting for twice its meansigma methods.Its fucoidan Bf demonstration as a result HGF produces induced activity.

1. 0 100 1 209 10 333 100 377 (condition is that the HGF generation of matched group is 9.17ng/ml to generation (%) (μ g/ml) sample of table 82 addition HGF.)

Embodiment 23

(1) ガ go メ Thallus Laminariae (Thallus Eckloniae) 500g is chopped up, after cleaning with 10 liter of 80% ethanol, stirred 3 days at 25 ℃ in 50 liters of 10% ethanol that contain 1mM potassium chloride, the stainless steel metal net filtration with mesh diameter 32 μ m obtains 45 liters of filtrates.34 liters of this filtrates after 3 hours, are cooled to 50 ℃ 80 ℃ of heating.It is concentrated when keeping 50 ℃ of liquid temperature with the ultrafiltration OMEGA box (Filtron society system) of getting rid of molecular weight 10,000.Further carry out desalination with heating to 50 ℃ distilled water 5L, adds same distilled water 200ml with loop washing 2 times, recovery obtains 1.5 liters of concentrated solutions.With its lyophilization, obtain 8.2g F-and be rich in fucoidan.

(2) with the same quadrat method of embodiment 1-(1), the HGF that measures the F-rich fucoidan (sample 1.) of embodiment 23-(1) preparation produces induced activity.Its result is shown in table 83.Its as a result F-be rich in fucoidan and show that HGF produces induced activity.

1. 0 100 1 117 10 186 100 244 (condition is that the HGF generation of matched group is 9.60ng/ml to generation (%) (μ g/ml) sample of table 83 addition HGF.)

Embodiment 24

To be suspended in the DMEM culture medium that contains 10% hyclone at the NHDF cell (people's normal skin fibroblast) of the DMEM culture medium culturing that contains 10% hyclone, reach 1 * 10 ⁵Cell/ml adds 48 porocyte culture plates with it, and every hole 500 μ l cultivated 24 hours.Afterwards, with the DMEM culture medium exchange that contains 1% hyclone, add minoxidil (with the pure pharmaceutical worker's industry of light society system) and the sample of 10 μ g/ml or 100 μ g/ml.In addition, in addition,, do not add minoxidil, only add sample, carry out equally as differentiation.Reclaim culture medium, use Quantikine human liver cell multiplicaiton factor (HGF) ELISA test kit, measure the HGF amount in the culture medium.Add the fucoidan Bf that embodiment 22 prepares respectively as sample, the F-of embodiment 23 preparations is rich in fucoidan, and the 7-12SFd-F of reference example 2-(3) preparation makes its ultimate density reach 1,10,100 μ g/ml respectively.In addition, also add the test that ultimate density is 0.1 μ g/ml for fucoidan Bf.As negative control, add distilled water with sample equivalent.Test is all carried out 3 times, gets its meansigma methods.The result is shown in table 84～86.

When not adding minoxidil, the HGF amount in the culture medium increases in the interdependent mode of concentration that fucoidan Bf, F-are rich in fucoidan, 7-12SFd-F, than there being the matched group that adds tangible increase is not arranged.Hence one can see that, even under the few situation of the mRNA of the HGF that resembles no minoxidil processing, also can promote synthetic two aspects free from the HGF of cell surface and HGF.In addition, when adding minoxidil, the HGF amount in the culture medium increases in the interdependent mode of concentration that fucoidan Bf, F-are rich in fucoidan, 7-12SFd-F, than there being the matched group that adds tangible increase is not arranged.In addition, the situation that the situation that such mRNA increases and the mRNA of no minoxidil processing are few is compared, and the increase of HGF is very significant.Hence one can see that, and fucoidan Bf, F-are rich in fucoidan, 7-12SFd-F is the free and generation that following of a spot of situation promotes HGF on a small quantity at mRNA, in addition, when mRNA exists in a large number, when promptly needing HGF in a large number, significantly promotes the free and generation of HGF.Concentration (pg/ml) the fucoidan Bf of HGF does not have minot ground minoxidil minoxidil (μ g/ml) that 10 μ g/ml 100 μ g/ml in the table 84 NHDF/ fucoidan Bf culture medium

0????????????N.D.???????126.04???????148.83

0.1??????????300.20?????307.00???????478.90

1????????????428.83?????448.17???????624.60

10???????????791.93?????799.90???????794.20

100 729.33 709.97 860.23N.D. represent to detect below the boundary line.Table 85 NHDF/F-is rich in the fucoidan culture medium HGF concentration (pg/ml) F-and is rich in fucoidan and does not have minoxidil minoxidil minoxidil minoxidil

(μG/ml)???????????????????1μg/ml????10μg/ml????100μg/Ml

0?????????????N.D.????????N.D.???????N.D.????????N.D.

1????????????132.75???????N.T.???????N.T.???????203.03

10???????????316.90??????239.90?????310.20??????434.10

100 369.33 327.70 372.70 537.87N.D. represent to detect below the boundary line.In addition, HGF concentration (pg/ml) 7-12SFd-F in the N.T. represents not estimate. table 86 NHDF/7-12SFd-F culture medium represents to detect below the boundary line without minoxidil minoxidil minoxidil (μ g/ml) 10 μ g/ml 100 μ g/ml 0 N.D. N.D. 137.20 1 228.73 164.00 378.30 10 345.93 376.07 450.83 100 439.67 483.20 820.10N.D..

Embodiment 25

(CDF1 is female 7 ages in week to mice, the about 20kg of body weight), use galactosamine (20mg/ mice) and LPS (lipopolysaccharide: 0.3 μ g/ mice) while intraperitoneal administration, manufacture the deadly model of acute severe hepatitis, the prolongation life effect of the fucoidan of research reference example 1-(1) record.Fucoidan is adjusted into 10% with distilled water, with the consumption of 10ml/kg body weight (fucoidan is 1g/kg), used galactosamine and LPS administration simultaneously preceding 1 hour and administration after carried out 2 times in 1 hour and force administration.Use same distilled water administration for matched group.

Survival rate after on-test after 72 hours is, 1 example of living in 8 examples in the matched group, and 7 examples of living in 8 examples in the fucoidan administration group, can confirm has significant life-saving effect by using the fucoidan administration.In addition, can confirm to survive and the serum biochemistry value is also had the effect of improvement in the example.Its result is shown in table 87.

Table 87 group GPT (U/l) GOT (U/l) total bilirubin

(mg/dl) contrast 385 613 1.0 fucoidan 94 ± 22 233 ± 53 0.3 ± 0.02 administration group

Embodiment 26

(1) ガ go メ Thallus Laminariae (Thallus Eckloniae) 500g is chopped up, after cleaning with 10 liter of 80% ethanol, in 50 liters of 10% ethanol that contain 1mM potassium chloride, in the container of internal diameter 40cm, stirred 2 days at 25 ℃, the speed of changeing with per minute 120 stirs, extract fucoidan, extract prepares fucoidan solution with the stainless steel metal net filtration of mesh diameter 32 μ m.

With 1g Petiolus Trachycarpi oil (Hua Wangshe system: used for cosmetic) be dissolved in 1 liter of ethanol, 1 liter of the Petiolus Trachycarpi oil solution of this preparation is joined when stirring in 46 liters of this fucoidan solution, add 1 liter of glycerol again, the preparation astringent.

(2) in the fucoidan solution of embodiment 26-(1) preparation, add gelatin and spice and reach ultimate density 0.02%, obtain using the astringent of gelatin.In addition, similarly add ossein, obtain using the astringent of ossein.

The biomaterial of preservation

(1) east, ripple city a kind in 1 fourth order No. 3 (postcode 305) is built in title address life Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce of preservation office Hitachinaka County, Japan

(2) microorganism of preservation

(i) replace zygosaccharomyces (Alteromonas sp.) SN-1009

Former preservation day: on February 13rd, 1996

Be transferred to international preservation request day: on November 15th, 1996

Accession designation number: FERM BP-5747

(ii) Flavobacterium (Flavobacterium sp.) SA-0082

Former preservation day: March 29 nineteen ninety-five

Be transferred to international preservation request day: on February 15th, 1996

Accession designation number: FERM BP-5402

Utilizability on the industry

The invention provides contain show growth factor produce material that induced activity gets as active ingredient for the effective medicine of disease that needs growth factor to produce. This medicine has HGF to produce induced activity in vivo, h-IGF produces induced activity, the NGF neurotrophic factor produces induced activity, and it is effective needing therapeutic agent or the prophylactic of the disease of these growth factors generations as hepatitis, diabetes, cancer, neurogenic disease etc.

In addition, use is selected from has the inducing acidic polysaccharose of growth factor, sulfated polysaccharides, for example fucose, dextran sulfate sodium, contain the extract of shark cartilage of chondroitin sulfate in a large number, its degradation product, acidic oligosaccharide, acid monose, and their material of salt can make the diet product, the growth factor that forms produces to induce with food, beverage and absorbs by the diet product, can improve the symptom of the disease that needs the growth factor generation. And can provide the feed with same physiological function.

Therefore, contain and be selected from that to have a HGF inducing, the acidic polysaccharose, sulfated polysaccharides, for example fucose that use in the present invention, the material of its degradation product, acidic oligosaccharide, acid monose and their salt is as the functional diet product of active ingredient, functional feed, because its growth factor produces inducing action, is functional diet product or feed useful in keeping the homeostasis of organism.

The present invention also provides HGF to produce to use biological cosmetics, and it is exceedingly useful in the health control of skin etc. In addition, also provide cancer transfer inhibitor.

In addition, also provide growth factor to produce derivant, this derivant the functional study of growth factor, with the growth factor relevant disease be useful in drug screening.

＜110〉＜120〉＜130〉00-028-PCT＜150〉JP 11-108067＜151〉1999-4-15＜150〉JP 11-108499＜151〉1999-4-15＜150〉JP 11-114542＜151〉1999-4-22＜150〉JP 11-129163＜151〉1999-5-10＜150〉JP 11-142343＜151〉1999-5-21＜150〉JP 11-154662＜151〉1999-6-2＜150〉JP 11-200982＜151〉1999-7-14＜150〉JP 11-275231＜151〉1999-9-28＜150〉JP 11-375606＜151〉1999-12-28＜150〉JP 2000-99941＜151〉2000-3-31＜160〉4＜210〉1＜211〉23＜212〉DNA＜213〉＜220〉＜223〉＜400〉1gtgaatactg cagaccaatg tgc 23＜210〉2＜211〉25＜212〉DNA＜213〉＜220〉＜223〉＜400〉2cacagacttc gtagcgtacc tctgg 25＜210〉3＜211〉21＜212〉DNA＜213〉＜220〉＜223〉＜400〉3caagagatgg ccacggctgc t 21＜210〉4＜211〉22＜212〉DNA＜213〉＜220〉＜223〉＜400〉4tccttctgca tcctgtcggc aa 22

Claims (32)

1. need somatomedin to produce inductive treatment of diseases agent or preventive, it is characterized in that containing be selected from have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and their material of salt (condition is, except the heparin, Heparan sulfate) as effective ingredient.

In the claim 1 record therapeutic agent or preventive, wherein, acidic polysaccharose is a sulfated polysaccharides.

In the claim 2 record therapeutic agent or preventive, wherein, sulfated polysaccharides is the sulfated polysaccharides of algae origin, the sulfated polysaccharides of animal origin, the sulfated polysaccharides of plant origin, the sulfated polysaccharides of microorganism origin or the sulfated polysaccharides of Fish origin.

4. the therapeutic agent or the preventive of record in the claim 2, wherein, sulfated polysaccharides is synthetic sulfated polysaccharides.

In the claim 2 record therapeutic agent or preventive, wherein, sulfated polysaccharides is a fucoidan.

In the claim 1 record therapeutic agent or preventive, wherein, acid monosaccharide is Glucose sulfate, sulphation galactose, sulphation xylose, sulphation 2-deoxyglucose, sulphation talose and sulphation mannose.

In the claim 1 record therapeutic agent or preventive, wherein, acidic oligosaccharide is to be selected from sulphation maltose, the sulphation lactose, sulphation sucrose, sulphation trehalose, the sulphation lactulose, the sulphation 6-(.alpha.-D-galactosido)-D-glucose., sulphation cellobiose, sulphation dextrinose, the sulphation turanose, sulphation パ ラ チ ノ one ス, sulphation maltotriose, Maltohexaose sulfate, sulphation Fructus Hordei Germinatus seven sugar, sulphation dodecyl MALTOHAXAOASE, the sulfated oligosaccharide of chemical compound shown in chemical compound shown in the following formula (I) and the following formula (II)

In the formula, R represents OH or OSO ₃H;

In the formula, R represents OH or OSO ₃H.

8. the therapeutic agent or the preventive of record in wantonly 1 of the claim 1～7, wherein, somatomedin is hepatocyte proliferation factor, Insulin-Like multiplicaiton factor or nerve growth factor.

9. the therapeutic agent or the preventive of record in wantonly 1 of the claim 1～8 is characterized in that further containing and can work in coordination with the inducing material of somatomedin that increases acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt.

In the claim 9 record therapeutic agent or preventive, wherein, can work in coordination with the inducing material of somatomedin that increases acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt is to be selected from cytokine class, prostaglandins and the material that contains the chemical compound of cyclopentenes ring.

11. contain be selected from have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and the somatomedin that forms of their material of salt produce and induce with food, beverage or feedstuff.

12. food, beverage or the feedstuff of record in the claim 11, wherein, acidic polysaccharose is a sulfated polysaccharides.

13. food, beverage or the feedstuff of record in the claim 12, wherein, sulfated polysaccharides is the sulfated polysaccharides of algae origin, the sulfated polysaccharides of animal origin, the sulfated polysaccharides of plant origin, the sulfated polysaccharides of microorganism origin or the sulfated polysaccharides of Fish origin.

14. food, beverage or the feedstuff of record in the claim 12, wherein, sulfated polysaccharides is synthetic sulfated polysaccharides.

15. food, beverage or the feedstuff of record in the claim 12, wherein, sulfated polysaccharides is a fucoidan.

16. food, beverage or the feedstuff of record in the claim 11, wherein, acid monosaccharide is Glucose sulfate, sulphation galactose, sulphation xylose, sulphation 2-deoxyglucose, sulphation talose and sulphation mannose.

17. food, beverage or the feedstuff of record in the claim 11, wherein, acidic oligosaccharide is to be selected from sulphation maltose, the sulphation lactose, sulphation sucrose, sulphation trehalose, the sulphation lactulose, the sulphation 6-(.alpha.-D-galactosido)-D-glucose., sulphation cellobiose, sulphation dextrinose, the sulphation turanose, sulphation パ ラ チ ノ one ス, sulphation maltotriose, Maltohexaose sulfate, sulphation Fructus Hordei Germinatus seven sugar, sulphation dodecyl MALTOHAXAOASE, the sulfated oligosaccharide of chemical compound shown in chemical compound shown in the following formula (I) and the following formula (II)

In the formula, R represents OH or OSO ₃H;

In the formula, R represents OH or OSO ₃H.

18. food, beverage or the feedstuff of record in wantonly 1 of the claim 11～17, its be hepatocyte proliferation factor produce induce usefulness, Insulin-Like multiplicaiton factor to produce to induce with or nerve growth factor production induce usefulness.

19. food, beverage or the feedstuff of record in wantonly 1 of the claim 11～18 is characterized in that further containing and can work in coordination with the inducing material of somatomedin that increases acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt.

20. food, beverage or the feedstuff of record in the claim 19, wherein, can work in coordination with the inducing material of somatomedin that increases acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt is to be selected from cytokine class, prostaglandins and the material that contains the chemical compound of cyclopentenes ring.

21. contain be selected from have the inducing acidic polysaccharose of somatomedin, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and the somatomedin made of their material of salt produce to induce and apply some make up.

22. the cosmetics of record in the claim 21, wherein, acidic polysaccharose is a sulfated polysaccharides.

23. the cosmetics of record in the claim 22, wherein, sulfated polysaccharides is the sulfated polysaccharides of algae origin, the sulfated polysaccharides of animal origin, the sulfated polysaccharides of plant origin, the sulfated polysaccharides of microorganism origin or the sulfated polysaccharides of Fish origin.

24. the cosmetics of record in the claim 22, wherein, sulfated polysaccharides is synthetic sulfated polysaccharides.

25. the cosmetics of record in the claim 22, wherein, sulfated polysaccharides is a fucoidan.

26. the cosmetics of record in the claim 21, wherein, acid monosaccharide is Glucose sulfate, sulphation galactose, sulphation xylose, sulphation 2-deoxyglucose, sulphation talose and sulphation mannose.

27. the cosmetics of record in the claim 21, wherein, acidic oligosaccharide is to be selected from sulphation maltose, the sulphation lactose, sulphation sucrose, sulphation trehalose, the sulphation lactulose, the sulphation 6-(.alpha.-D-galactosido)-D-glucose., sulphation cellobiose, sulphation dextrinose, the sulphation turanose, sulphation パ ラ チ ノ one ス, sulphation maltotriose, Maltohexaose sulfate, sulphation Fructus Hordei Germinatus seven sugar, sulphation dodecyl MALTOHAXAOASE, the sulfated oligosaccharide of chemical compound shown in chemical compound shown in the following formula (I) and the following formula (II)

In the formula, R represents OH or OSO ₃H;

In the formula, R represents OH or OSO ₃H.

28. the cosmetics of record in wantonly 1 of the claim 21～27, its induce for hepatocyte proliferation factor produces usefulness, the generation of Insulin-Like multiplicaiton factor induce with or nerve growth factor production induce and apply some make up.

29. the cosmetics of record in wantonly 1 of the claim 21～28 is characterized in that further containing and can work in coordination with the inducing material of somatomedin that increases acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt.

30. the cosmetics of record in the claim 29, wherein, can work in coordination with the inducing material of somatomedin that increases acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol or their salt is to be selected from cytokine class, prostaglandins and the material that contains the chemical compound of cyclopentenes ring.

31. the cosmetics of record in wantonly 1 of the claim 21～30, it is lotion class, emulsion class, cream kind, facial film class, baths, and the agent of washing one's face is bathed with soap or bath lotion.

32. somatomedin produces regulator, it is characterized in that containing be selected from acidic polysaccharose, its degradation product, acidic oligosaccharide, acid monosaccharide, acid sugar alcohol, and their material of salt as effective ingredient.